Adhesin tip multiepitope fusion antigen platform and vaccines and their use in the treatment of enterotoxigenic diarrhea

ABSTRACT

The application provides for MEFA constructs and vaccines against Entertoxigenic  Escherichia coli  (ETEC). Methods for reducing the incidence of diarrhea associated with ETEC are also provided. The representative adhesin tip MEFAs and the representative major subunit CFA MEFA-II provided in the disclosure has an adhesin tip backbone or major subunit CFA with one or more adhesin tips, adhesin subunit or major structural subunit epitopes incorporated onto the backbone. The adhesin tip MEFAs and the CFA MEFA-II provided advantageously prevents the ETEC molecule from attaching to the intestine.

BACKGROUND OF THE INVENTION

Diarrhea continues to be a leading cause of death in children younger than 5 years who living in developing countries. Enterotoxigenic Escherichia coli (ETEC) is a leading bacterial cause of children's diarrhea and travelers' diarrhea. ETEC bacteria attaching host receptors at epithelial cells and colonizing in the small intestine initiate ETEC-associated diarrheal disease. Therefore, preventing ETEC attachment has been considered the first line of defense against ETEC diarrhea. However, ETEC strains produce over 23 immunologically heterogeneous adhesins, which makes developing vaccines that broadly protect against ETEC bacterial attachment challenging. Accordingly, what is needed is a composition that provides a protective effect against ETEC. What is further needed is a composition that induces antibody responses to multiple ETEC adhesins. What is also needed is a composition that impairs, reduces, or even prevents ETEC attachment.

SUMMARY OF THE INVENTION

The present disclosure overcomes the problems inherent in the prior art by applying a MEFA (multiepitope fusion antigen) approach to integrate epitopes from adhesin tips or adhesive subunits of CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21, EtpA, or other ETEC adhesins and to construct representative adhesin tip MEFA peptides (tip MEFA-I, tip MEFA-II), or epitopes from the major structural subunits of CS7, CS12, CS14, CS17, CS19, CS21, EtpA, and EaeH adhesins for a major subunit CFA MEFA (CFA MEFA-II). The antigenicity of each tip MEFA or the CFA MEFA was then examined in mouse immunization, and assessed for potential application for ETEC vaccine development. Data showed mice intraperitoneally immunized with ETEC adhesin tip MEFA-I developed IgG antibody responses to all nine ETEC adhesins (CFA/I, CS1-CS6, CS21, EtpA). Moreover, ETEC and E. coli bacteria expressing these nine adhesins, after incubation with serum of the immunized mice, had significant reduction of attachment to Caco-2 cells. Similarly, mice immunized with ETEC adhesin tip MEFA-II developed IgG antibodies against attachment of CS12, CS14, CS17, CS19 and EaeH, and mice immunized with major subunit CFA MEFA-II developed IgG antibodies against CS7, CS12, CS14, CS17, CS19, CS21, EtpA and EaeH adhesins. These results indicated that anti-adhesin antibodies induced by an adhesin tip MEFA or a CFA major subunit MEFA blocked adherence of the most important ETEC adhesins, suggesting that multivalent tip MEFA-I, tip MEFA-II, and the CFA MEFA-II can be useful for developing a broadly protective anti-adhesin vaccine against ETEC diarrhea.

Thus, the present disclosure utilizes an MEFA (multiepitope fusion antigen) approach to integrate epitopes from adhesin tips or adhesive subunits of CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21 CS7, CS12, CS14, CS17, CS19, EaeH, and EtpA adhesins to construct at least one adhesin tip MEFA peptide(s). Data showed mice intraperitoneally or subcutaneously immunized with an adhesin tip MEFA developed IgG antibody responses to all representing ETEC adhesins. Moreover, after incubation with serum of the immunized mice, ETEC and E. coli bacteria expressing these adhesins had significant reduction of attachment to Caco-2 cells. This MEFA approach was also used to integrate epitopes from the major structural subunits of CS7, CS12, CS14, CS17, CS19, CS21, EtpA and EaeH adhesins into a CFA major structural subunit MEFA (CFA MEFA-II). This CFA MEFA-II was then examined for immunogenicity in mouse immunization studies. Data showed that immunized mice developed strong IgG antibodies to each of eight target adhesins; moreover, the induced antibodies significantly reduced adherence of ETEC strains expressing each of these eight adhesins to Caco-2 cells. These results indicated that anti-adhesin antibodies induced by each adhesin tip MEFA or the CFA MEFA-II blocked adherence of the most important ETEC adhesins, suggesting the multivalent tip MEFA (tip MEFA-I, tip MEFA-II) or the major structural subunit MEFA (CFA MEFA-II) can be useful for developing a broadly protective anti-adhesin vaccine against ETEC diarrhea. “MEFA” refers to and encompasses both adhesin tip and major structural subunit MEFA.

The present disclosure provides for at least three, preferably, four, more preferably five, still more preferably six, even more preferably seven, still more preferably eight or more multi epitope fusion antigens (MEFA) comprising a nucleotide fragment encoding a polypeptide backbone from a colonization factor antigen (CFA) having epitopes from adhesin tips, adhesive subunits or major structural subunits of Enterotoxigenic Escherichia coli (ETEC) incorporated therein. In one embodiment, the epitopes from adhesin tips or adhesive subunits of ETEC are preferably selected from the group consisting of CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS12, CS14, CS17, CS19, CS21, EtpA, EaeH, and any combination thereof. In one embodiment, there may be a single or multiple copies of each epitope present in the MEFA from the structural major subunits of CS7, CS12, CS14, CS17, CS19, CS21, EtpA and EaeH adhesins present in a MEFA.

In a further aspect, the present disclosure provides for an adhesin tip MEFA comprising nucleotides encoding a polypeptide backbone from a colonization factor antigen (CFA) having epitopes from adhesin tips or adhesive subunits of ETEC incorporated therein, wherein the backbone to which the epitopes are attached is preferably selected from, but not limited to, CfaE, CS14, and CS21. The present disclosure also provides for an adhesin major structural subunit CFA MEFA-II composing nucleotides encoding a polypeptide backbone from a colonization factor antigen (CFA) having epitopes from adhesin major structural subunits of CS7, CS12, CS14, CS17, CS19, CS21, EtpA, and EaeH ETEC adhesins incorporated therein.

The adhesin tip or major subunit MEFAs disclosed herein may further comprise additional elements, where such elements are selected from the group consisting of, but not limited to, heat liable toxin (LT), heat stable toxin (STa), an epitope from adhesin of enteroaggregative E. coli (EAEC), epitopes of cholera, epitopes of rotavirus, and any combination thereof. There may be one or more copies of LT and/or STa in any given adhesin tip MEFA of the present disclosure.

In one aspect, the present disclosure provides for a nucleic acid molecule that encodes for the polypeptide molecules described herein.

Further, the present disclosure provides for an expression vector comprising a nucleic acid molecule encoding the polypeptide molecules described herein.

In another aspect, the present disclosure provides for an immunogenic composition or vaccine composition comprising the polypeptide molecules described herein and a pharmaceutically acceptable vehicle, adjuvant, carrier and any combination thereof.

In a further aspect, a method of inducing an immune response against ETEC is provided. Preferably, the steps of the method include, but are not limited to, administration of one or more of the polypeptide(s) described herein to a human or animal in need thereof.

In yet another aspect, a method of reducing the severity or incidence of the clinical signs of ETEC infection is also provided in the present disclosure. The method of reducing the clinical signs of ETEC infection includes, but is not limited to, the step of administration of one or more the polypeptide(s) and/or adhesin tip MEFA(s) and/or CFA major subunit MEFA(s) disclosed herein to a human or animal in need thereof. The reduction in severity or incidence is in comparison to an animal or human, or group of animals or humans, that has not received an administration of one or more polypeptide(s) of the present disclosure.

Another aspect of the present disclosure includes a method of treating or reducing the incidence or severity of diarrhea. The method preferably comprises the steps of administration of one or more of the polypeptide(s) and/or adhesin tip MEFA(s) and/or CFA major subunit MEFA(s), as described herein, to a human or animal in need thereof. The treating or reducing the incidence or severity of diarrhea is in comparison to an animal or human, or group of animals or humans, that has not received an administration of one or more polypeptide(s) and/or adhesin tip MEFA(s) and/or CFA major subunit MEFA(s) of the present disclosure.

A method of reducing adherence of the ETEC bacteria to the intestine is also provided. The method preferably comprises the steps of administration of one or more of the polypeptide(s) and/or adhesion tip MEFA(s) and/or CFA major subunit MEFA(s) to a human or animal in need thereof. The adherence is in comparison to an animal or human, or group of animals or humans, that has not received an administration of one or more polypeptide(s) and/or adhesin tip MEFA(s) and/or CFA major subunit MEFA(s) of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings described herein are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

FIG. 1 is a schematic illustration of the constructed ETEC adhesin tip MEFA-I protein. Amino acid sequences of the epitopes from each adhesin tip or adhesive subunit are shown in the top panel. The bottom left panel shows results of Western blot with mouse anti-CFA/I antiserum, 9450 was the adhesin tip MEFA protein (25 and 30 μl), and (−) was the total proteins from BL21 E. coli. The bottom right panel showed Coomassie blue staining of refolded solubilized adhesin tip MEFA proteins (lane 1: protein marker; lane 2: 25 μl tip MEFA protein; lane 3: 30 μl tip MEFA protein);

FIG. 2 is a plot of serum anti-CfaE (CFA/I), -CooD (CS1), -CotD (CS2), -CstH (CS3), -CsaE (CS4), -CsfD (CS5), -CssB (CS6), anti-LngA (CS21) and anti-EtpA (EtpA) IgG antibody titers (log₁₀) from mice immunized with tip MEFA-I. Solid circles (.) are titers of mice intraperitoneally immunized with the adhesin tip MEFA-I protein. Each circle represented the IgG titer from an individual mouse. Bars indicated the mean titers of the group specific to each adhesin;

FIG. 3 is an illustration of a host cell and bacterial cell showing adhesin interaction;

FIG. 4 is an illustration of the genetic structure of adhesin MEFA-I with a CS14 adhesin tip subunit CsuD as the backbone, with two CsuD epitopes retained, where 4 epitopes were substituted with epitopes from tip subunits of CS12 (CswF), CS17 (CsbD), CS19 (CsdD) and EaeH adhesins;

FIG. 5 is a plot of serum IgG antibody titers (log₁₀) for anti-CS12, anti-CS14, anti CS17, anti-CS19, and anti-EaeH IgG antibodies from mice subcutaneously immunized with adhesin tip MEFA-II protein, where each dot represented an IgG titer from an individual mouse and bars indicated mean titers;

FIG. 6 is an illustration of the genetic structure of major subunit CFA MEFA-II with a CS21 major subunit LngA as the backbone, with a LngA surface-exposed and epitopes from the major subunits of CS7, CS12, CS14, CS17, CS19, EtpA and EaeH adhesins;

FIG. 7 is a plot of serum IgG antibody titers (log₁₀) for anti-CS7, anti-CS12, anti-CS14, anti CS17, anti-CS19, anti-CS21, anti-EaeH and anti-EtpA IgG antibodies from mice subcutaneously immunized with major subunit CFA MEFA-II, where each dot represented an IgG titer from an individual mouse and bars indicated mean titers; and

FIG. 8 is an illustration of a MEFA of the present disclosure illustrating the epitopes being incorporated into the backbone.

DETAILED DESCRIPTION

The present disclosure provides for a multi epitope fusion antigen (MEFA) comprising a polypeptide including epitopes from adhesin tips or adhesive subunits of Enterotoxigenic Escherichia coli (ETEC). In one embodiment, the epitopes from adhesin tips or adhesive subunits of ETEC are preferably selected from the group consisting of the adhesin tip or adhesive subunit of CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS12, CS14, CS17, CS19, CS21, EtpA, EaeH and combinations thereof. In one embodiment, the polypeptide comprises at least 2 epitopes, at least 3 epitopes, at least 4 epitopes, at least 5 epitopes, at least 6 epitopes, at least 7 epitopes, at least 8 epitopes, at least 9 epitopes, at least 10 epitopes, at least 11 epitopes, at least 12 epitopes, at least 13 epitopes, at least 14 epitopes, and at least 15 epitopes.

The CS1 epitope for purposes of the present disclosure includes any nucleotide sequence or the amino acid sequence encoded by a nucleotide sequence that is associated with the CS1 genome from ETEC, including any fragment or portion thereof. The CS1 genome is preferably the nucleotide sequence encoding SEQ ID No.47 or any sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No. 47. In one embodiment, the CS1 epitope nucleotide sequence is generated from the primers selected from SEQ ID No. 3 and SEQ ID No. 4. One such preferred nucleotide sequence is SEQ ID No. 55. In an alternate embodiment, the CS1 epitope nucleotide sequence is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with a sequence generated from the primers selected from SEQ ID No. 3 and SEQ ID No. 4. Preferably, the CS1 epitope is an adhesin tip or adhesive subunit. In one embodiment, the epitope from CS1 is selected from the nucleotide sequence encoding SEQ ID No. 37 and a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with the nucleotide sequence encoding SEQ ID No. 37.

The CS2 epitope for purposes of the present disclosure includes any nucleotide sequence or the amino acid sequence encoded by the nucleotide sequence that is associated with the CS2 genome from ETEC, including any fragment or portion thereof. The CS2 genome is preferably the nucleotide sequence encoding SEQ ID No.48 or any sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No. 48. In one embodiment, the CS2 epitope nucleotide sequence is generated from the primers selected from SEQ ID No. 5 and SEQ ID No. 6. One such preferred nucleotide sequence is SEQ ID No. 56. In an alternate embodiment, the CS2 epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with a sequence generated from the primers selected from SEQ ID No. 5 and SEQ ID No. 6. Preferably, the CS2 epitope is an adhesin tip or adhesive subunit. In one embodiment, the epitope from CS2 is selected from the nucleotide sequence encoding SEQ ID No. 42 and a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with the nucleotide sequence encoding SEQ ID No. 42.

The CS3 epitope for purposes of the present disclosure includes any nucleotide sequence or the amino acid sequence encoded by the nucleotide sequence that is associated with the CS3 genome from ETEC, including any fragment or portion thereof. The CS3 genome is preferably the nucleotide sequence encoding SEQ ID No.49 or any sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No. 49. In one embodiment, the CS3 epitope nucleotide sequence is generated from the primers selected from SEQ ID No. 7 and SEQ ID No. 8. One such preferred nucleotide sequence is SEQ ID No. 57. In an alternate embodiment, the CS3 epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with a sequence generated from the primers selected from SEQ ID No. 7 and SEQ ID No. 8. Preferably, the CS3 epitope is an adhesin tip or adhesive subunit. In one embodiment, the epitope from CS3 is selected from the nucleotide sequence encoding SEQ ID No. 36 and a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with the nucleotide sequence encoding SEQ ID No. 36.

The CS4 epitope for purposes of the present disclosure includes any nucleotide sequence or the amino acid sequence encoded by the nucleotide sequence that is associated with the CS4 genome from ETEC, including any fragment or portion thereof. The CS4 genome is preferably the nucleotide sequence encoding SEQ ID No.50 or any sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No. 50. In one embodiment, the CS4 epitope nucleotide sequence is generated from the primers selected from SEQ ID No. 9 and SEQ ID No. 10. One such preferred nucleotide sequence is SEQ ID No. 58. In an alternate embodiment, the CS4 epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with a sequence generated from the primers selected from SEQ ID No. 9 and SEQ ID No. 10. Preferably, the CS4 epitope is an adhesin tip or adhesive subunit. In one embodiment, the epitope from CS4 is selected from the nucleotide sequence encoding SEQ ID No. 41 and a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with the nucleotide sequence encoding SEQ ID No. 41.

The CS5 epitope for purposes of the present disclosure includes any nucleotide sequence or the amino acid sequence encoded by the nucleotide sequence that is associated with the CS5 genome from ETEC, including any fragment or portion thereof. The CS5 genome is preferably the nucleotide sequence encoding SEQ ID No.51 or any sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No. 51. In one embodiment, the CS5 epitope nucleotide sequence is generated from the primers selected from SEQ ID No. 11 and SEQ ID No. 12. One such preferred nucleotide sequence is SEQ ID No. 59. In an alternate embodiment, the CS5 epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with a sequence generated from the primers selected from SEQ ID No. 11 and SEQ ID No. 12. Preferably, the CS5 epitope is an adhesin tip or adhesive subunit. In one embodiment, the epitope from CS5 is selected from the nucleotide sequence encoding SEQ ID No. 35 and a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with the nucleotide sequence encoding SEQ ID No. 35.

The CS6 epitope for purposes of the present disclosure includes any nucleotide sequence or the amino acid sequence encoded by the nucleotide sequence that is associated with the CS6 genome from ETEC, including any fragment or portion thereof. The CS6 genome is preferably the nucleotide sequence encoding SEQ ID No.52 or any sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No. 52. In one embodiment, the CS6 epitope nucleotide sequence is generated from the primers selected from SEQ ID No. 15 and SEQ ID No. 16. One such preferred nucleotide sequence is SEQ ID No. 60 or any sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No. 60.

In an alternate embodiment, the CS6 epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with a sequence generated from the primers selected from SEQ ID No. 13 and SEQ ID No. 14. Preferably, the CS6 epitope is an adhesin tip or adhesive subunit. In one embodiment, the epitope from CS6 is selected from the nucleotide sequence encoding SEQ ID No. 38 and a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with the nucleotide sequence encoding SEQ ID No. 38.

The CS21 epitope for purposes of the present disclosure includes any nucleotide sequence or the amino acid sequence encoded by the nucleotide sequence that is associated with the CS21 genome from ETEC, including any fragment or portion thereof. The CS21 genome is preferably the nucleotide sequence encoding SEQ ID No.53 or any sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No. 53. In one embodiment, the CS21 epitope nucleotide sequence is generated from the primers selected from SEQ ID No. 15 and SEQ ID No. 16. One such preferred nucleotide sequence is SEQ ID No. 61. In an alternate embodiment, the CS21 epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with a sequence generated from the primers selected from SEQ ID No. 15 and SEQ ID No. 16. Preferably, the CS21 epitope is an adhesin tip or adhesive subunit. In one embodiment, the epitope from CS21 is selected from the nucleotide sequence encoding SEQ ID No. 39 and a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with the nucleotide sequence encoding SEQ ID No. 39.

The EtpA epitope for purposes of the present disclosure includes any nucleotide sequence or the amino acid sequence encoded by the nucleotide sequence that is associated with the EtpA genome from ETEC, including any fragment or portion thereof. The EtpA genome is preferably the nucleotide sequence encoding SEQ ID No.2 or any sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No. 2. In one embodiment, the EtpA epitope nucleotide sequence is generated from the primers selected from SEQ ID No. 17 and SEQ ID No. 34. One such preferred nucleotide sequence is SEQ ID No. 62. In an alternate embodiment, the EtpA epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with a sequence generated from the primers selected from SEQ ID No. 17 and SEQ ID No. 34. Preferably, the EtpA epitope is an adhesin tip or adhesive subunit. In one embodiment, the epitope from EtpA is selected from the nucleotide sequence encoding SEQ ID No. 40 and a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with the nucleotide sequence encoding SEQ ID No. 40.

The CS7 epitope for purposes of the present disclosure includes any nucleotide sequence or the amino acid sequence encoded by the nucleotide sequence that is associated with the CS7 genome from ETEC, including any fragment or portion thereof. In one embodiment, the CS7 epitope is selected from the nucleic acid sequence encoding SEQ ID No. 22 and/or the nucleic acid sequence encoding SEQ ID No. 29 and/or the nucleic acid sequence encoding SEQ Id NO. 68. In an alternate embodiment, the CS7 epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with either the nucleic acid sequence encoding SEQ ID No. 22 and/or the nucleic acid sequence encoding SEQ ID No. 29 and/or the nucleic acid sequence encoding SEQ Id NO. 68. In another alternate embodiment, the CS7 epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No. 22 and/or SEQ ID No. 29 and/or the nucleic acid sequence encoding SEQ Id NO. 68. Preferably, the CS7 epitope is an adhesin tip or adhesive subunit.

The CS12 epitope for purposes of the present disclosure includes any nucleotide sequence or the amino acid sequence encoded by the nucleotide sequence that is associated with the CS12 genome from ETEC, including any fragment or portion thereof. In one embodiment, the CS12 epitope is selected from the nucleic acid sequence encoding SEQ ID No. 24 and/or the nucleic acid sequence encoding SEQ ID No. 30 and/or the nucleic acid sequence encoding SEQ Id NO. 69. In an alternate embodiment, the CS12 epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with either the nucleic acid sequence encoding SEQ ID No. 24 or the nucleic acid sequence encoding SEQ ID No. 30 and/or the nucleic acid sequence encoding SEQ Id NO. 69. In another alternate embodiment, the CS12 epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No. 24 and/or SEQ ID No. 30 and/or the nucleic acid sequence encoding SEQ Id NO. 69. Preferably, the CS12 epitope is an adhesin tip or adhesive subunit.

The CS14 epitope for purposes of the present disclosure includes any nucleotide sequence or the amino acid sequence encoded by the nucleotide sequence that is associated with the CS14 genome from ETEC, including any fragment or portion thereof. In one embodiment, the CS14 epitope is selected from the nucleic acid sequence encoding SEQ ID No. 21, and/or the nucleic acid sequence encoding SEQ ID No. 23, and/or the nucleic acid sequence encoding SEQ ID No. 26 and/or the nucleic acid sequence encoding SEQ Id NO. 70. In an alternate embodiment, the CS14 epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with one or more of the nucleic acid sequence encoding SEQ ID No. 21, the nucleic acid sequence encoding SEQ ID No. 23, and the nucleic acid sequence encoding SEQ ID No. 26 and/or the nucleic acid sequence encoding SEQ Id NO. 70. In another alternate embodiment, the CS14 epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No. 21, SEQ ID No. 23, and/or SEQ ID No. 26 and/or the nucleic acid sequence encoding SEQ Id NO. 70. Preferably, the CS14 epitope is an adhesin tip or adhesive subunit.

The CS17 epitope for purposes of the present disclosure includes any nucleotide sequence or the amino acid sequence encoded by the nucleotide sequence that is associated with the CS17 genome from ETEC, including any fragment or portion thereof. In one embodiment, the CS17 epitope is selected from the nucleotide sequence encoding SEQ ID No. 25 and/or the nucleotide sequence encoding SEQ ID No. 31 and/or the nucleic acid sequence encoding SEQ ID NO. 71. In an alternate embodiment, the CS17 epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with either the nucleotide sequence encoding SEQ ID No. 25 or the nucleotide sequence encoding SEQ ID No. 31 and/or the nucleic acid sequence encoding SEQ ID NO. 71. In another alternate embodiment, the CS17 epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No. 25 and/or SEQ ID No. 31 and/or the nucleic acid sequence encoding SEQ ID NO. 71. Preferably, the CS17 epitope is an adhesin tip or adhesive subunit.

The CS19 epitope for purposes of the present disclosure includes any nucleotide sequence or the amino acid sequence encoded by the nucleotide sequence that is associated with the CS19 genome from ETEC, including any fragment or portion thereof. In one embodiment, the CS19 epitope is selected from the nucleotide sequence encoding SEQ ID No. 27 and/or the nucleotide sequence encoding SEQ ID No. 32 and/or the nucleic acid sequence encoding SEQ ID NO. 72. In an alternate embodiment, the CS19 epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with either the nucleotide sequence encoding SEQ ID No. 27 or the nucleotide sequence encoding SEQ ID No. 32 and/or the nucleic acid sequence encoding SEQ ID NO. 72. In another alternate embodiment, the CS19 epitope is selected from SEQ ID No. 27 and/or SEQ ID No. 32 and/or the nucleic acid sequence encoding SEQ ID NO. 72. Preferably, the CS19 epitope is an adhesin tip or adhesive subunit.

The EaeH epitope for purposes of the present disclosure includes any nucleotide sequence or the amino acid sequence encoded by the nucleotide sequence that is associated with the EaeH genome from ETEC, including any fragment or portion thereof. In one embodiment, the EaeH epitope is selected from the nucleotide sequence encoding SEQ ID No. 28 and/or the nucleotide sequence encoding SEQ ID No. 33 and/or the nucleic acid sequence encoding SEQ ID NO. 73. In an alternate embodiment, the EaeH epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with either the nucleotide sequence encoding SEQ ID No. 28 and/or the nucleotide sequence encoding SEQ ID No. 33 and/or the nucleic acid sequence encoding SEQ ID NO. 73. In an alternate embodiment, the EaeH epitope is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No. 28 and/or SEQ ID No. 33 and/or the nucleic acid sequence encoding SEQ ID NO. 73. Preferably, the EaeH epitope is an adhesin tip or adhesive subunit.

In a further aspect, the present disclosure provides for an adhesin tip MEFA comprising a polypeptide encoding epitopes from adhesion tips or adhesive subunits of ETEC, wherein the polypeptide encodes a colonization factor antigen (CFA) backbone to which the epitopes are attached. The backbone is preferably selected from, but not limited to, CfaE, CfaA, CfaB, CfaC, CS14, CS7, CS12, CS17, CS19, CS21, and combinations thereof. In a particularly preferred embodiment, the backbone is selected from CfaE, CS14, and CS21.

The CfaE backbone for purposes of the present disclosure includes any nucleotide sequence or amino acid sequence or the product produced by the nucleotide sequence associated with the CfaE genome from ETEC, including any fragment or portion thereof. In one embodiment, the CfaE backbone is selected from the sequence generated by the primers SEQ ID No. 18 and SEQ ID No. 19. In an alternate embodiment, the CfaE backbone is selected from a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with a sequence generated by the primers SEQ ID No. 18 and SEQ ID No. 19. In some preferred forms, the CfaE backbone has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No. 46. In another embodiment, the amino acid sequence of the CfaE backbone is SEQ ID No. 46. In a further embodiment, the CfaE backbone retains one or more epitopes of CfaE. Such CfaE epitopes are preferably selected from the nucleotide sequence encoding SEQ ID No. 43, the nucleotide sequence encoding SEQ ID No. 44, the nucleotide sequence encoding SEQ ID No. 45, and sequences having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with the nucleotide sequence encoding SEQ ID No. 43, SEQ ID NO. 44, or SEQ ID No. 45.

In a further aspect, the present disclosure provides for an adhesin major subunit MEFA of CS21 comprising a polypeptide encoding epitopes from adhesion tips or adhesive subunits of CS7, CS12, CS14, CS17, CS19, EtpA, and EaeH, wherein the polypeptide encodes a colonization factor antigen (CFA) backbone to which the epitopes are attached. In a preferred aspect, the CS21 backbone retains two epitopes.

The CS14 backbone for purposes of the present disclosure includes any nucleotide sequence or amino acid sequence or the product produced by the nucleotide sequence associated with the CS14 genome from ETEC, including any fragment or portion thereof. In one embodiment, the CS14 backbone is selected from the nucleotide sequence encoding SEQ ID No. 21 and a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with the nucleotide sequence encoding SEQ ID No.21. In an alternative embodiment, the CS14 backbone is selected from the a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No.21. In a further embodiment, the CS14 backbone retains one or more epitopes of CS14. Such CS14 epitopes are preferably selected from the nucleotide sequence encoding SEQ ID No. 23, the nucleotide sequence encoding SEQ ID No. 26, and sequences having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with the nucleotide sequence encoding SEQ ID No. 23 or SEQ ID No. 26.

The CS7 backbone for purposes of the present disclosure includes any nucleotide sequence or amino acid sequence that is associated with the CS7 genome from ETEC, including any fragment or portion thereof. In one embodiment, the CS7 backbone is selected from the nucleotide sequence encoding SEQ ID No. 29 and/or a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with a nucleotide sequence encoding SEQ ID No.29. In an alternative embodiment, the CS7 backbone is selected from the a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No.29.

The CS12 backbone for purposes of the present disclosure includes any nucleotide sequence or amino acid sequence that is associated with the CS12 genome from ETEC, including any fragment or portion thereof. In one embodiment, the CS12 backbone is selected from the nucleotide sequence encoding SEQ ID No. 30 and/or a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with the nucleotide sequence encoding SEQ ID No.30. In an alternative embodiment, the CS12 backbone is selected from the a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No.30.

The CS17 backbone for purposes of the present disclosure includes any nucleotide sequence or amino acid that is associated with the CS17 genome from ETEC, including any fragment or portion thereof. In one embodiment, the CS17 backbone is selected from the nucleotide sequence encoding SEQ ID No. 31 and/or a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with the nucleotide sequence encoding SEQ ID No.31. In an alternative embodiment, the CS17 backbone is selected from the a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No. 31.

The CS19 backbone for purposes of the present disclosure includes any nucleotide sequence or amino acid sequence that is associated with the CS19 genome from ETEC, including any fragment or portion thereof. In one embodiment, the CS19 backbone is selected from the nucleotide sequence encoding SEQ ID No. 21 and/or a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with the nucleotide sequence encoding SEQ ID No.32. In an alternative embodiment, the CS19 backbone is selected from the a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with SEQ ID No. 32.

The adhesin tip MEFAs disclosed herein may further comprise additional elements, where such elements are selected from, but not limited to, heat liable toxin (LT), heat stable toxin (STa), epitopes from adhesin of enteroaggregative E. coli (EAEC), epitopes of cholera, epitopes of rotavirus and any combination thereof. In one embodiment, there may be one or more copies of LT, STa, and any combination thereof.

For purposes of the present invention, the adhesin tip MEFA may be comprised of any nucleotide encoding the backbone of a CFA from ETEC having at least one of the epitopes from ETEC inserted therein. One non-limiting example of such an adhesin tip MEFA is the nucleotide sequence of SEQ ID No. 1 or the amino acid sequence of SEQ ID No. 20, however, the disclosure is not so limited as those of skill in the art can appreciate that many combinations are possible. In one embodiment, the complete adhesin tip MEFA of the present invention is a sequence that is at least 80%, at least 85% at least 90%, at least 95%, or 100% identical to the nucleotide sequence of SEQ ID No. 1 or the amino acid sequence of SEQ ID No. 20. In another embodiment, the complete adhesin tip MEFA of the present invention is a sequence that is at least 80%, at least 85% at least 90%, at least 95%, or 100% identical to the nucleotide sequence of SEQ ID No. 63 or the amino acid sequence of SEQ ID No. 64. In another embodiment, the complete adhesin tip MEFA of the present invention is a sequence that is at least 80%, at least 85% at least 90%, at least 95%, or 100% identical to the nucleotide sequence of SEQ ID No. 65 or the amino acid sequence of SEQ ID No. 66.

In one aspect, the present disclosure provides for a nucleic acid molecule that encodes for the polypeptide molecules described herein.

Further, the present disclosure provides for an expression vector comprising the nucleic acid molecule encoding the polypeptide molecules described herein. The expression vector is preferably selected from, but not limited to, E. coli, Salmonella, and combinations thereof.

The E. coli preferably used for purposes of the present disclosure is selected from, but not limited to, H10407, THK/38/pEU405, DH5α/pEU588. E116 (E19446), E106 (E11881/9), UM 75688, JF2423 ETP98066, JF2101, JF2318 ETP050008, CFA/I knockout, 9573, 9474, 9475, 9505, 9504, 9533, 9506, 9468, 9507, 9450, and any combination thereof.

In another aspect, the present disclosure provides for an immunogenic composition or vaccine composition comprising one or more of the adhesin tip MEFAs encoding polypeptide molecules described herein and a pharmaceutically acceptable vehicle. The one or more polypeptide molecules may be multiple copies of the same polypeptide or more than one polypeptide, each having a different structure. It is appreciated that there are many combinations of adhesin tip MEFAs encoding polypeptide molecules that may be present in an immunogenic composition or vaccine.

In one embodiment, the immunogenic composition or vaccine disclosed herein comprises an adhesin tip MEFA comprising a polypeptide encoding for a backbone, preferably the CfaE backbone, having the epitopes for CS1, CS2, CS3, CS4, CS5, CS6, CS21, and EtpA.

In a further embodiment, the immunogenic composition or vaccine disclosed herein comprises an adhesin tip MEFA comprising a polypeptide encoding for a backbone, preferably the CS14 backbone, having the epitopes for CS7, CS12, CS17, CS14, CS19, and EaeH.

In yet another embodiment, the immunogenic composition or vaccine disclosed herein comprises two adhesin tip MEFAs. It is appreciated that any combination of a plurality of backbones with different epitopes thereon is contemplated in the present disclosure. In one preferred embodiment, the first adhesin tip MEFA comprises a polypeptide encoding for a CfaE backbone having the epitopes for CS1, CS2, CS3, CS4, CS5, CS6, CS21, and EtpA; and the second adhesin tip MEFA comprises a polypeptide encoding for a CS14 backbone having the epitopes for CS7, CS12, CS17, CS14, CS19, and EaeH.

The immunogenic composition of the present disclosure may further comprise a preservative, stabilizer, carrier, adjuvant, and/or pharmaceutical vehicle. “Adjuvants” as used herein, can include aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.), water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion. The emulsion can be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil such as squalane or squalene; oil resulting from theoligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di-(caprylate/caprate), glyceryl tri-(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, D. E. S.). John Wiley and Sons, NY, pp 51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997).

For example, it is possible to use the SPT emulsion described on page 147 of “Vaccine Design, The Subunit and Adjuvant Approach” edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of this same book.

A further instance of an adjuvant is a compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative. Advantageous adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U.S. Pat. No. 2,909,462 which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name Carbopol; (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with an allyl sucrose or with allyl pentaerythritol. Among then, there may be mentioned Carbopol 974P, 934P and 971P. Most preferred is the use of Cabopol 971P. Among the copolymers of maleic anhydride and alkenyl derivative, the copolymers EMA (Monsanto) which are copolymers of maleic anhydride and ethylene. The dissolution of these polymers in water leads to an acid solution that will be neutralized, preferably to physiological pH, in order to give the adjuvant solution into which the immunogenic, immunological or vaccine composition itself will be incorporated.

Further suitable adjuvants include, but are not limited to, the RIM adjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta Ga.), SAF-M (Chiron, Emeryville Calif.), monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, IMS 1314 or muramyl dipeptide among many others. Most preferably, heat-labile enterotoxin mutants are used as the adjuvant.

In a further aspect, a method of inducing an immune response against ETEC is provided. Preferably, the steps of the method include, but are not limited to, administration of one or more of the polypeptide(s) described herein to a human or animal in need thereof.

A method of reducing the incidence of or severity of clinical signs of ETEC infection is also provided in the present disclosure. The method of reducing the incidence or severity clinical signs of ETEC infection includes, but is not limited to, the step of administration of one or more the polypeptide(s) disclosed herein to a human or animal in need thereof. In one embodiment, the severity or incidence of clinical signs of ETEC are reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and 100% when compared to a human or animal, or group of humans or animals not having received the adhesin tip MEFA. The MEFA is preferably administered in an amount of less than 50 micrograms, where values such as 1-30 micrograms, 10-30 micrograms, 10-20 micrograms, 10-50 micrograms, 20-50 micrograms, 30-50 micrograms, 40-50 micrograms, 1 microgram, 5 micrograms, 10 micrograms, 15 micrograms, 20 micrograms, 25 micrograms, 30 micrograms, 35 micrograms, 40 micrograms, 45 micrograms, and 50 micrograms are envisioned. The route of administration is preferably selected from intradermal, intramuscular, or subcutaneous immunization.

A method of treating or reducing the incidence or severity of diarrhea is also provided in the present disclosure. The method preferably comprises the steps of administration of one or more of the adhesin tip MEFA(s) of the present disclosure to a human or animal, or group of humans or animals in need thereof. In one embodiment, diarrhea incidence or severity is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and 100% when compared to a human or animal, or group of humans or animals not having received the administration of the adhesin tip MEFA.

A method of reducing the adhesion of ETEC to the gut is also provided by the present disclosure. The method preferably comprises the steps of administration of one of more of the adhesin tip MEFA(s) of the present disclosure to a human or animal in need thereof. In one embodiment, adhesion to the gut is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and 100% when compared to a human or animal not having received the adhesin tip MEFA

As used herein “multiepitope fusion antigen” (MEFA) refers to a molecule that is created by combining the fusion and epitope strategies for making vaccines or immunogenic compositions. The method of producing an MEFA allows for use of a backbone molecule and the addition of any antigenic element or identified virulence factors, as well as additional elements. In the present disclosure, the MEFA may have a single copy or multiple copies of any one component. The “adhesin tip MEFA” means that the MEFA is created using the “tip” or end of the genome or a subunit that is on the “tip” of the gene.

The “adhesin tip” for purposes of the present disclosure refers to a subunit protein located at the tip of an ETEC bacteria adhesion.

For purposes of the present disclosure, “adhesive subunit” refers to a subunit protein of ETEC adhesion which is not located at the tip, but is involved in bacterial adherence to host receptors.

For purposes of the present disclosure, “sequence identity” refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position-by-position basis, e.g., the sequences are “identical” at a particular position if at that position, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give % sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences.

For purposes of the present disclosure, “sequence homology refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned, and gaps are introduced if necessary. However, in contrast to “sequence identity”, conservative amino acid substitutions are counted as a match when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology with a reference sequence, 85%, preferably 90%, even more preferably 95% of the amino acid residues or nucleotides in the reference sequence must match or comprise a conservative substitution with another amino acid or nucleotide, or a number of amino acids or nucleotides up to 15%, preferably up to 10%, even more preferably up to 5% of the total amino acid residues or nucleotides, not including conservative substitutions, in the reference sequence may be inserted into the reference sequence.

For purposes of the present disclosure, “epitope” refers to a short peptide which functions as an antigen molecule to induce antibody response.

For purposes of the present disclosure, “backbone” refers to a protein that serves as a support of the molecule to maintain a stable protein structure and also to present foreign epitopes of interest.

The MEFA of the present invention is not a nucleotide or amino acid sequence found in nature, as it has been constructed by the hand of man. Therefore, the MEFA of the present invention is markedly different from what is found in nature. Similar to Example 5 for the Nature-Based Product Examples of eligible subject matter under 35 U.S.C. 101 issued by the US Patent Office in 2014, the MEFA of the present invention is like claim 2 of that example because the MEFA gene has additional elements, such as the epitopes that provides it with a functionally different characteristic than naturally occurring ETEC bacteria. Most notably, the MEFA of the present invention fails to adhere to the intestine, which is a marked difference from ETEC bacteria found in nature.

All ranges provided herein include each and every value in the range as well as all sub-ranges there-in-between as if each such value or sub-range was disclosed. Further, all aspects and embodiments of the disclosure comprise, consist essentially of, or consist of any aspect or embodiment, or combination of aspects and embodiments disclosed herein.

EXAMPLES

The following examples are simply intended to further illustrate and explain the present invention. The invention, therefore, should not be limited to any of the details in these examples.

Example 1

Materials and Methods

Bacterial strains and plasmids: E. coli and ETEC strains used as DNA templates for PCR amplification of the adhesin tip or adhesive subunit genes of CFA/I, CS1-CS6, CS21 and EtpA adhesins and for in vitro antibody adherence inhibition assay, as well as recombinant E. coli strains expressing each adhesin tip, adhesive subunit protein and the tip MEFA-I protein are listed below in Table 1. Plasmid pET28α (Novagen, Madison, Wis.) and E. coli strain BL21 (GE Healthcare, Piscataway, N.J.) were used to express adhesin tips, adhesive subunits, and the adhesin tip MEFA-I protein.

TABLE 1 A list of Escherichia coli strains used in this study. E. coli and ETEC strains were used as templates for amplification of adhesin tip genes, and also used in antibody adherence inhibition assay. Recombinant E. coli strains expressing each adhesin tip constructed in this study were also included. Strain Relevant characteristics Source H10407 O78:H11; CFA/I, LT, STa Johns Hopkins University THK38/pEU405 CS1 Emory University DH5α/pEU588 CS2 Emory University E116 (E19446) CS3, LT, STa University of Gothenburg E106 (E11881/9) CS4/CS6, LT, STa University of Gothenburg UM 75688 CS5/CS6, LT, STa Johns Hopkins University JF2423 ETP98066 CS6, LT, STa Washington University JF2101 CS21, EtpA, STa Washington University JF2318 ETP050008 EtpA, STa Washington University CFA/I knockout ΔCFA/I H10407, EtpA, University of LT, STa Maryland 9473 CfaE (CFA/I) in this study pET28α/BL21 Kan⁺ 9474 CooD (CS1) in this study pET28α/BL21 Kan⁺ 9475 CotD (CS2) in this study pET28α/BL21 Kan⁺ 9505 CstH (CS3) in this study pET28α/BL21 Kan⁺ 9504 CsaE (CS4) in this study pET28α/BL21 Kan⁺ 9533 CsfD (CS5) in this study pET28α/BL21 Kan⁺ 9506 CssB (CS6) in this study pET28α/BL21 Kan⁺ 9468 LngA (CS21) in this study pET28α/BL21 Kan⁺ 9507 EtpA in this study pET28α/BL21 Kan⁺ 9450 adhesin tip MEFA-I in this study pET28α/BL21 Kan⁺

Cloning and expression of adhesin tips, adhesive subunits and adhesin tip MEFA-I: PCR primers used to amplify the adhesin tip genes of CFA/I, CS1, CS2, CS3, CS4 and CS5, and adhesive subunit (major structural subunit involving in adhesion) genes of CS6, CS21 and EtpA, are listed in Table 2. PCR amplified adhesin tip or adhesive subunit gene products were digested with restriction enzymes EagI and NcoI or NheI (New England BioLabs Inc, Ipswich, Mass.). Digested products were cloned into vector pET28α and expressed in E. coli strain BL21.

TABLE 2 PCR primers used to amplify adhesin tip subunits in this study. Restriction sites, NheI or NcoI in forward primers and EagI in reverse primers, are underlined. Primer Sequence (5′→3′) CfaE-F CTAGCTAGCGATAAAAATCCCGGAAGTG (SEQ ID No. 18) CfaE-R GATCGGCCGCTAGAGTGTTTGACTACTTG (SEQ ID No. 19) CS1 (CooD)-F CTAGCTAGCGGGCGATACCCGGAAACTACAG (SEQ ID No. 3) CS1 (CooD)-R GATCGGCCGTCATAAATTTTCGACACTGG (SEQ ID No. 4) CS2 (CotD)-F CTAGCTAGCCAATCATGGCATACGAACGTAG (SEQ ID No. 5) CS2 (CotD)-R GATCGGCCGTTACAGACTTGAACTACTAGGAG (SEQ ID No. 6) CS3 (CstH)-F AGTTACATCCATGGGCACTCTAACCAAAGAACTGGCATTAAATGTGC  (SEQ ID No. 7) CS3 (CstH)-R TACATGATCGGCCGTTAATTACCTGAAACTAAATGTTCGTTACC (SEQ ID No. 8) CS4 (CsaE)-F CTAGCTAGCGATAAAATTCCCGGAGATGAAAG (SEQ ID No. 9) CS4 (CsaE)-R GATCGGCCGCTAGAGTGTTTGACTACTTGGTGTG (SEQ ID No. 10) CS5 (CsfD)-F TTTTCCATGGTTATGGTTCAGGCTGCTACA (SEQ ID No. 11) CS5 (CsfD)-R AGATCGGCCGTTATTTATTGTAACATTTCC (SEQ ID No. 12) CS6 (CssB)-F AGTTACATCCATGGGCTGGCAATATAAATCTCTGGATGTAAATG (SEQ ID No. 13) CS6 (CssB)-R ATGTAGATCGGCCGTTAAGTCAAATTTCCTGCATAAGTACCAGAC (SEQ ID No. 14) CS21 (LngA)-F CTAGCTAGCATGAGCCTGCTGGAAGTTATCATTG (SEQ ID No. 15) CS21 (LngA)-R GATCGGCCGTTAACGGCTACCTAAAGTAATTG (SEQ ID No. 16) EtpA-F CTAGCTAGCGGCGTGGGTAATGCAAAAGCCACG (SEQ ID No. 17) EtpA-R ATGTAGATCGGCCGTTAGCTGAAGGTGTAACGACGGTTCATG (SEQ ID No. 34)

To construct the adhesin tip MEFA-I gene, first web-based B-cell epitope prediction software was used to in silico identify B-cell epitopes from the adhesin tips of the six adhesins (CFA/I, CS1-CS5) and adhesive subunits of three adhesins (CS6, CS21, EtpA). Then the CFA/I tip CfaE gene (cfaE) was applied as backbone, then eight nucleotide fragments coding surface-exposed but less antigenic peptides were truncated, as confirmed by DNA sequencing, and the nucleotide fragments coding the most antigenic epitope of the other eight adhesin tips or adhesive subunits were inserted into the truncated positions of the cfaE gene. After in silico optimization of epitope presentation, this adhesin tip MEFA-I gene was synthesized (gene synthesis performed by Genewiz, South Plainfield, N.J.). The synthetic gene was digested with enzymes NcoI and EagI, and were cloned in vector pET28α and expressed in E. coli BL21.

A single colony from each recombinant strain was cultured in 5 ml Luria-Bertani (LB) broth supplemented with kanamycin (30 μg/ml) at 37° C. overnight. Overnight grown culture was added to 200 ml 2× YT medium (2× Yeast Extract Tryptone). After optical density (OD) reached 0.6, bacteria were induced with 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG; Sigma, St. Louis, Mo.) and grew for four more hours. Bacteria were collected with centrifugation of 12,000-13,000 rpm for 15 minutes. Pellets, after frozen and thaw, were suspended in 10 ml bacterial protein extraction reagent (B-PER; Pierce, Rockford, Ill.) with vortex. After 30 min at room temperature, bacteria lysates were centrifuged at 12,000-13,000 rpm for 15 min at 4° C. Pellets were suspended in 10-20 ml B-PER with vigorous vortex and pipetting with a 20-ml syringe, mixed with 200-400 μl freshly prepared lysozyme (10 mg/ml, in B-PER), and incubated at room temperature for 20 min. Suspensions were centrifuged; resultant pellets were suspended and then incubated with lysozyme again. After centrifugation, pellets were suspended in 100 ml 1:10 diluted B-PER, vortexed, and centrifuged. Pellets were washed three times with 100 ml PBS, vortexed vigorously, centrifuged at 12,000 rpm for 15 min at 4° C. Final pellets were dissolved in 10 ml PBS.

Extracted proteins were then refolded using a protein refolding kit by following manufacturer's protocol (Novagen). Refolded solubilized proteins were transferred to molecular porous membrane tubing (Spectrum Laboratories, Inc., Rancho Dominguez, Calif.), and dialyzed in 20 mM Tris-HCl, pH8.5 (supplemented with 0.1 mM DTT) at 4° C. After 3-4 h, protein samples were moved to dialysis buffer without DTT, and followed by two more buffer changes. Solubilized proteins (in supernatant) were collected with centrifugation at 12,000 rpm for 10 min at 4° C., and were stored at −80° C.

Mouse immunization with adhesin tip MEFA-I was performed by the following steps. Before being used in mouse immunization, 25 or 30 μl refolded solubilized adhesin tip MEFA-I protein samples (1 mg/ml) were examined in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with mouse anti-CFA/I antiserum (1:3000). IRDye-labeled goat anti-mouse IgG (1:5000; LI-COR, Lincoln, Nebr.) was used as the secondary antibody. LI-COR Odyssey premium infrared gel imaging system was used to visualize bound proteins. In addition, Coomassie blue staining was used to assess purity of the extracted adhesin tip MEFA-I proteins.

Seven-week-old female BALB/c mice (Charlies River Laboratories International, Inc., Wilmington, Mass.) were used in mouse immunization. A group of eight mice were each intraperitoneally immunized with 150 μg (in 200 μl PBS) adhesin tip MEFA-I protein generated above, together with 1 μg dmLT (LT_(R192G/L211A); provided by Walter Reed Army Institute of Research, Silver Spring, Md.) as adjuvant. Immunized mice received two booster injections intraperitoneally at the same dosage amount of the primary, in an interval of two weeks. Two weeks after the second booster, mice were sacrificed. A group of eight mice without immunization were served as the control group.

Serum samples were collected from each mouse before the primary and two weeks after the final booster, and were stored at −80° C. until use. The mouse immunization study complied with the American Welfare Act by following the 1996 National Research Council guidance and was approved by the Kansas State University's Institutional Animal Care and Use Committee.

Mouse anti-CFA IgG antibody titration was performed by the following steps. Recombinant adhesin tip and adhesive subunit proteins were used as coating antigens respectively in ELISAs to titrate mouse serum anti-CfaE (CFA/I), -CooD (CS1), -CotD (CS2), -CstH (CS3), -CsaE (CS4), -CsfD (CS5), -CssB (CS6), anti-LngA (CS21) and anti-EtpA IgG antibodies in this study. Serum samples collected from each mouse prior to the primary immunization and two weeks after the final booster were initially diluted in 1:400 and then two-folded diluted till to 1:51,200. Four hundred nanogram of each adhesin tip or subunit recombinant protein, in 100 μl coating buffer (41), was added to each well of Immulon 2HB 96-well plates (Thermo Fisher Scientific, Rochester, N.Y.). Plates were incubated at 37° C. for 1 h and then 4° C. overnight. Coated plates were washed three times with PBS-0.05% Tween 20 (PBST), and uncoated sites were blocked with 5% nonfat milk at 37° C. for 1 h. After washing three times with PBST, wells were incubated with mouse serum dilutions at 37° C. for 1 hour. After washing 3 times with PBST, wells were incubated with horseradish peroxidase (HRP) conjugated goat-anti-mouse IgG antibodies (1:3000; Sigma) at 37° c. for 1 h. After 3 washes with PBST and 2 washes with PBS, wells were incubated with 100 μl 3,3′,5,5′-tetramethylbezidine (TMB) Microwell Peroxidase Substrate (KPL, Gaithersburg, Md.) at room temperature for 30 min, and were measured for optical density (OD) at 650-nm wavelength. OD readings from the highest dilution that gave readings above 0.3 after subtraction of background readings (OD×dilution) were calculated to antibody titers and presented in log₁₀ scale.

A mouse serum antibody adherence inhibition assay was performed by the following steps. Mouse serum in vitro antibody adherence inhibition assay was carried out. Briefly, 30 μl serum pooled from the immunized mice or the control mice was added to ETEC or E. coli bacteria (4.5×10⁶ CFUs; in 150 μl PBS) that express CFA/I, CS1, CS2, CS3, CS4/CS6, CS5/CS6, CS6, CS21, or EtpA. Incubated at room temperature for 1 h on a shaker (50 rpm), the serum/bacteria mixture was brought to 300 μl with PBS and added to Caco-2 cells (3×10⁵ cells, in 700 μl DMEM-10% FBS; at a multiplicity-of-infection ratio of 15 bacteria to 1 cell). After incubation at 37° C. for 1 h in a CO₂ incubator (5% CO₂), Caco-2 cells were gently washed 3 times with PBS to remove non-adherent bacteria, and then dislodged with 0.5% triton X (300₁1.1 per well; 37° C. for 20 min at room temperature). Adherent bacteria (and dislodged Caco-2 cells) were collected with centrifugation, suspended in 1 ml PBS, serially diluted and plated on LB plates. Plates were cultured at 37° C. overnight, and counted for CFUs.

Statistical analysis: Data of antibody adherence inhibition assay were analyzed using the mixed procedure (SAS for windows, version 8; SAS Institute, Cary, N.C.). Results were expressed in means±standard deviations. Student's t-test was used to compare differences between the immunization group and the control group. Differences were considered significant if p<0.05 when treatments were compared at two-tailed distribution and two-sample unequal variance.

Results and Conclusions

Adhesin tip MEFA-I protein carried epitopes of nine adhesin tips or subunits. Epitopes ‘QFTEKRSLIKR’ (CS1/SEQ ID No. 37), SQSIEMRFQDDSQ′ (CS2/SEQ ID No. 42), ‘NITLDKNAGNT’ (CS3/SEQ ID No. 36), ‘FYLRKINDDTK’ (CS4/SEQ ID No. 41), ‘DIGRLQSDAEY’ (CS5/SEQ ID No. 35), ‘YDSDPKLDSQ’ (CS6/SEQ ID No. 38), ‘VAMKDAYQRDGKYPDF’ (CS21/SEQ ID No. 39), and ‘VEGEKSPRRGV’ (EtpA/SEQ ID No. 40) were in silico predicted from CooD of CS1, CotD of CS2, CstH of CS3, CsaE of CS4, CsfD of CS5, CssB of CS6, LngA of CS21 and EtpA of EtpA, respectively. Three epitopes from CfaE of CFA/I: TEKRSLIKRELQIK′ (SEQ ID No. 43), ‘QDDNSKSDGKFYLKKINDDSKE’ (SEQ ID No. 44) and ‘RLQLDAKVKNPEA’ (SEQ ID No. 45) were retained in the tip MEFA-I (FIG. 1). This 362-amino acid polypeptide was expressed, extracted, and recognized by mouse anti-CFA/I antiserum (FIG. 1).

It was found that mice intraperitoneally immunized with the adhesin tip MEFA-I developed immune responses to all nine adhesins. All eight mice intraperitoneally immunized with the adhesin tip MEFA-I recombinant protein developed antibody responses to each of the nine adhesin tips or adhesive subunits (FIG. 2). Anti-CfaE, -CooD, -CotD, -CstH, -CsaE, -CsfD, -CssB, and -LngA and anti-EtpA IgG antibody titers in the immunized mice were 3.1±0.36, 3.8±0.10, 3.2±0.19, 3.5±0.14, 3.8±0.13, 3.5±0.19, 4.0±0.16, 3.9±0.04 and 4.0±0.13 (in log₁₀), respectively. No antibody titers to these adhesin tip or subunit antigens were detected in the serum samples of the control mice, or serum collected before the primary immunization.

It was further discovered that mouse serum antibodies inhibited adherence of ETEC or E. coli bacteria expressing these nine adhesins. ETEC bacteria expressing CFA/I, CS3, CS4/CS6, CS5/CS6, CS6, CS21 or EtpA, or H10407 CfaE knock out mutant which expresses EtpA, and recombinant E. coli strains expressing CS1 or CS2 showed significant reduction in adherence to Caco-2 cells, after being incubated with the pooled serum sample of the immunized mice, compared to the same bacteria incubated with the pooled serum of the control mice.

Discussion

The adhesin tip MEFA-I created from this study carried epitopes from nine different ETEC adhesins and induced antibody responses to each of these nine adhesins. That may rejuvenate the concept of epitope vaccinology for developing safe and broadly protective vaccines against immunologically heterogeneous pathogens. Differing from the conceptual epitope vaccine strategy that stacks multiple epitopes into a linear peptide antigen and often leads to inferior immunogenicity or protection, the present disclosure applied the MEFA (multiepitope fusion antigen) approach to use a backbone protein and to substitute surface-exposed peptides of the backbone protein with epitopes of foreign antigens. This MEFA approach results in a protein which better presents the antigenic epitopes of interest. Future protein structural studies will help to understand antigenic topology of this MEFA-I protein.

The present disclosure included a CssB epitope to represent CS6 adhesin in this tip MEFA construction. Both CssA and CssB are the major structural subunits and play key roles in CS6 adherence. CssA epitope ‘⁷²QVTVYPV⁷⁸’ (SEQ ID No. 54) was found capable of inducing in vitro protective antibodies against CS6 adhesin adherence. Data from this study showed that the CssB epitope ‘YDSDPKLDSQ’ (SEQ ID No. 52) also induced antibodies that inhibited CS6 adherence to Caco-2 cells.

CstH is the major structural subunit and also the adhesive subunit for CS3 adhesin. CstH epitope ‘NITLDKNAGNT’ (SEQ ID No. 49) in this tip MEFA-I was able to induce strong anti-CstH antibody response. Comparison of antibody titers derived from the major subunit MEFA-I and this tip MEFA-I may not be much informative since different coating antigens were used in antibody titration ELISAs. In vitro antibody adherence inhibition assays suggested antibodies derived against these two CstH epitopes showed similar rates (50% vs. 58% from anti-CFA MEFA-I antibodies and anti-adhesin tip MEFA-I antibodies) inhibiting adherence of CS3 adhesin to Caco-2 cells. Future studies to identify optimal epitopes for ETEC adhesin tips or major subunits will help to improve efficacy of vaccine candidates.

Results from this study showed that the adhesin tip MEFA-I carried adhesin tip or adhesive subunit epitopes induced antibody responses to nine ETEC adhesins, and derived antibodies inhibited adherence of E. coli and ETEC bacteria expressing these nine adhesins to Caco-2 cells. Future studies using different immunization routes, different animals and challenge studies will be needed to further characterize antigen antigenicity and potential vaccine application against ETEC diarrhea.

Example 2

Materials and Methods

The method described above will be completed for another embodiment of the present disclosure, an adhesin tip MEFA (tip MEFA-I utilizing the backbone of CS14 having epitopes for CS7, CS12, CS17, CS14, CS19, and EaEH.

TABLE 3 This table shows the sequences of the complete MEFA-II sequence, as well as its constituent elements. Adhesin Tip MEFA-II (final product for 6 adhesins) (SEQ ID No. 20) MNKILFIFTLFFSSVLFTFAVSAQEGSSNRAKIDQTGDYTNIFGPRDRNESSPKHNILNDYI TAYSESHTLYDRMIFLCLSSQNTLNGACPTSENPSSSSVSGETNITLQFTEKRSLIKRELQI KGYKRLLFKGANCPSYLTLNSAHYNKDGEGVSPGGASLYLYIPAGELKNLPFGGIWDAT LKLRVKRRYDQTYGTYTINITVKLTDKGNIQIWLPQFKSDARVDLNLSFQAPRQDRSVQ SGRNSVDMCFYDGYSTNSSSLELRFQDNNPKSDGKFYLRKINDDTKEIAYTLSLLLAGK SAGNKKPIWENQSCDNIADAASLEINWNRITAVTMPEISVPVLCWPGRLQLDAKKSPDIE DYQERPANGYMGNINITFTPSSQTL Note: underlined amino acids are the epitopes from the tips of 6 adhesins. CS14-CsuD Backbone: CS14 Amino Acid (386) (SEQ ID No., 21) MNKILFIFTLFFSSVLFTFAVSADKIPGDENITNIFGPRDRNESSPKHNILNDYITAYSESHT LYDRMIFLCLSSQNTLNGACPTSENPSSSSVSGETNITLQFTEKRSLIKRELQIKGYKRLLF KGANCPSYLTLNSAHYTCNRNSASGASLYLYIPAGELKNLPFGGIWDATLKLRVKRRYD QTYGTYTINITVKLTDKGNIQIWLPQFKSDARVDLNLRPTGGGTYIGRNSVDMCFYDGY STNSSSLELRFQDNNPKSDGKFYLRKINDDTKEIAYTLSLLLAGKSLTPTNGTSLNIADAA SLEINWNRITAVTMPEISVPVLCWPGRLQLDAKVENPEAGQYMGNINITFTPSSQTL Epitopes predicted: 23-33, 84-99, 141-150, 221-231, 255-264, 289-299, 336-346. Replacement Epitopes substituted for predicted epitopes above. CS7: QEGSSNRAKIDQTGDY 24-39 (SEQ ID No. 22) CS14: PTSENPSSSSVSGETN 91-106 (retained from the backbone) (SEQ ID No. 23) CS12: NKDGEGVSPG 149-158 (SEQ ID No. 24) CS17: SFQAPRQDRSVQS 231-243 (SEQ ID No. 25) CS14: QDNNPKSDGK; 268-277 (SEQ ID No. 26) CS19: AGNKKPIWENQSCD 303-316 (SEQ ID No. 27) EaEH: KKSPDIEDYQERPANG 355-370 (SEQ ID No. 28) CS14 tip CSuD Backbone Nucleotide sequence (SEQ ID NO. 109) ATGAATAAGATTTTATTTATTTTTACATTGTTTTTCTCTTCAGTACTTTTTACATTTGC TGTATCGGCAGATAAAATTCCCGGAGATGAGAATATAACTAATATTTTTGGCCCGCG TGACAGGAACGAATCTTCCCCCAAACATAATATATTAAATGACTATATTACAGCATA CAGTGAAAGTCATACTCTGTATGATAGGATGATTTTTTTATGTTTGTCTTCTCAAAAT ACACTTAATGGAGCATGTCCAACCAGTGAGAATCCTAGCAGTTCATCGGTCAGTGGC GAAACAAATATAACATTACAATTTACGGAAAAAAGAAGTTTAATTAAAAGAGAGCT ACAAATTAAAGGCTATAAACGATTATTGTTCAAAGGTGCTAACTGCCCATCCTACCT AACACTTAACTCAGCTCATTATACCTGCAATAGAAACTCGGCTTCAGGTGCAAGTTT ATATTTATATATTCCTGCTGGCGAACTAAAAAATTTACCTTTTGGTGGTATCTGGGAT GCTACTCTGAAGTTAAGAGTAAAAAGACGATATGATCAGACCTATGGAACTTACAC TATAAATATCACTGTTAAATTAACTGATAAGGGAAATATTCAGATATGGTTACCTCA GTTCAAAAGTGACGCTCGCGTCGATCTTAACTTGCGTCCAACTGGTGGGGGCACATA TATTGGAAGAAATTCTGTTGATATGTGCTTTTATGATGGATATAGTACTAACAGCAG CTCTTTGGAGCTAAGATTTCAGGATAACAATCCTAAATCTGATGGGAAATTTTATCT AAGGAAAATAAATGATGACACCAAAGAAATTGCATATACTTTGTCACTTCTCTTGGC GGGTAAAAGTTTAACTCCAACAAATGGAACGTCATTAAATATTGCTGACGCAGCTTC TCTGGAAATAAACTGGAATAGAATTACAGCTGTCACCATGCCAGAAATCAGTGTTCC GGTGTTGTGTTGGCCTGGACGTTTGCAATTGGATGCAAAAGTGGAAAATCCCGAGGC CGGACAATATATGGGTAATATTAATATTACTTTCACACCAAGTAGTCAAACACTCTA G CS7-Cs213 (SEQ ID No. 29) MKNKLLFMMLTILGAPGIAAAAGYDLANSEYNFAVNELSKSSFNQAAIIGQAGTNNSAQ LRQGGSKLLAVVAQEGSSNRAKIDQTGDYNLAYIDQAGSANDASISQGAYGNTAMIIQK GSGNKANITQYGTQKTAIVVQRQSQMAIRVTQR Epitope [73-88 (16)]: QEGSSNRAKIDQTGDY (SEQ ID No. 22) CS12-CswF (SEQ ID No. 30) MLKRISCIIFVFFSGLIYAAEITNQIELSVKVNISKPMCKLNSGTQTIDEGDFDVLDIITENR KLNGHATFKFTECSSVKNMKIKEKQAGQNPALDIVNNYIPNSKGDRMAKGVAVKLLDD KKQEIQLNKEMNVIVEESLTEKDLTLNAQVISINKDGEGVSPGLLQTAIGMEISYE Epitope (155-164): NKDGEGVSPG (SEQ ID No. 24) CS12-CswF Nucleotide Sequence (SEQ ID No. 110) atgttgaaaag aatatcgtgt ataatttttg tttttttttc agggctgatt tatgctgcgg aaattacaaa tcagatagag ctttcggtaa aggttaatat atctaagcct atgtgtaaac ttaattctgg aacgcaaaca atagacttcg gcgattttga tgtactggat attattacgg agaacagaaa attaaatggt catgcgacct ttaaatttac tgagtgtagt tctgtcaaaa acatgaagat aaaatttaaa caggcaggac aaaatccagc gttagatatc gtaaacaatt atatccctaa tagtaaggga gatagaatgg caaagggggt agcggtaaag cttctggatg ataaaaagca agaaattcaa ctgaacaagg aaatgaatgt tattgtggag gagagtctga catttaaaga tttaacgtta aatgctcagg ttatctctat taataaagac ggagagggag tttcacctgg gctacttcag accgcaatag gaatggagat atcctatgaa tga Primers to amplify the segment for coating antigen in antibody titration ELISA RN-CS12-F: ATA TCC ATG GCT ATG TGT AAA CTT AAT TCT GGA (SEQ ID No. 114) RN-CS12-R: ATC TCG GCC GCT ATT GCG GTC TGA AGT AGC (SEQ ID No. 115) CS17-CsbD (SEQ ID No. 31) MKKIFIFLSIIFSAVVSAGRYPETTVGNLTKSFQAPRQDRSVQSPIYNIFTNHVAGYSLSHN LYDRIVELCTSSSNPVNGACPTLGTSGVQYGTTTITLQFTEKRSLIKRNINLAGNKKPIWE NQSCDTSNLMVLNSKSWSCGHYGNANGTLLNLYIPAGEINKLPFGGIWEATLILRLSRY GEVSSTHYGNYTVNITVDLTDKGNIQVWLPGEHSNPRVDLNLHPIGNYKYSGSNSLDMC FYDGYSTNSDSMVIKFQDDNPTYSSEYNLYKIGGTEKLPYAVSLLMGEKIFYPVNGQSFT INDSSVLETNWNRVTAVAMPEVNVPVLCWPARLLLNADVNAPDAGQYSGQIYITFTPS VENL Epitope (32-44): SFQAPRQDRSVQS (SEQ ID No. 25) CS17 CsbD Nucleotide Sequence (SEQ ID No. 111) atgaaaa agatatttat ttttttgtct atcatatttt ctgcggtggt cagtgccggg cgatacccgg aaactacagt aggtaatctg acgaagagtt ttcaagcccc tcgtcaggat agaagcgtac aatcaccaat atataacatc tttacgaatc atgtggctgg atatagtttg agtcataact tatatgacag gattgttttt ttatgtacat cctcgtcgaa tccggttaat ggtgcttgcc caacccttgg aacatctgga gttcaatacg gtactacaac cataaccttg cagtttacag aaaaaagaag tctgataaaa agaaatatta atcttgcagg taataagaaa ccaatatggg agaatcagag ttgcgacact agcaatctaa tggtgttgaa ttcgaagtct tggtcctgtg ggcattacgg aaatgctaac ggaacacttc taaatctgta tatccctgca ggagaaatca acaaattgcc ttttggaggg atatgggagg caactctgat cttacgctta tcaagatatg gcgaagtcag tagcacccat tacggcaatt ataccgtaaa tattacggtt gatttaactg ataaaggtaa tattcaggta tggcttccag ggtttcacag caacccgcgt gtagacctga atctgcaccc tatcggtaat tataaatata gtggtagtaa ttcactcgac atgtgtttct atgatggata tagtacaaac agtgatagca tggtaataaa gttccaggat gataatccta cctattcatc tgaatataat ctttataaga tagggggcac tgaaaaatta ccatatgctg tttcactgct tatgggagaa aaaatatttt atccagtgaa tggtcaatca tttactatca atgacagtag tgtactcgaa acaaactgga atcgagtaac cgcagttgct atgccggaag ttaatgttcc agtattatgc tggccagcaa gattgctatt aaatgctgat gtaaatgctc ccgatgcagg acagtattca ggacagatat ataacatt tacacccagt gtcgaaaatt tatga Primers to amplify the segment for coating antigen in antibody titration ELISA RN-CS17-F: TTT TCC ATG GGC ATG AAA AAG ATA TTT ATT TTT (SEQ ID No. 116) RN-CS17-R: GGT GCG GCC GTT ATA TAT ATC TGT CCT GAA (SEQ ID No. 117) CS19-CsdD (SEQ ID No. 32) MKKIFIFLSIIFSAVVSAGRYPETTVGNLTKSFQAPRLDRSVQSPIYNIFTNEIVAGYSLSHR LYDRIVFVCTSSSNPVNGACPTIGTSRVEYGTTTITLQFTEKRSLIKRNINLAGNKKPIWEN QSCDTSNLMVLNSKSWSCGALGNANGTLLNLYIPAGEINKLPFGGIWEATLILRLSRYGE VSSTHYGNYTVNITVDLTDKGNIQVWLPGFHSNPRVDLNLHPIGNYKYSGSNSLDMCFY DGYSTNSDSMVIKFQDDNPTNSSEYNTLYKIGGTEKLPYAVSLLMGGKIFYPVNGQSFTIN DSSVLETNWNRVTAVAMPEVNPVLCWPARLLLNADVNAPDAGQYSGQIYITFTPSVE NL Epitope (115-128): AGNKKPIWENQSCD (SEQ ID No. 27) C519-CsdD (SEQ ID No. 112) Nucleotide Sequence atgaaaaa gatatttatt tttttgtcta tcatattttc tgcggtggtc agtgccgggc gatacccgga aactacagta ggtaatctga cgaagagttt tcaagcccct cgtctggata gaagcgtaca atcaccaata tataacatct ttacgaatca tgtggctgga tatagtttga gtcatagatt atatgacagg attgtttttg tatgtacatc ctcgtcgaat ccggttaatg gtgcttgccc aaccattgga acatctagag ttgaatacgg tactacaacc ataaccttgc agtttacaga aaaaagaagt ctgataaaaa gaaatattaa tcttgcaggt aataagaaac caatatggga gaatcagagt tgcgacacta gcaatctaat ggtgttgaat tcgaagtctt ggtcctgtgg ggctctagga aatgctaacg gaacacttct aaatctgtat atccctgcag gagaaatcaa caaattgcct tttggaggga tatgggaggc aactctgatc ttacgcttat caagatatgg cgaagtcagt agcacccatt acggcaatta taccgtaaat attacggttg atttaactga taaaggtaat attcaggtat ggcttccagg gtttcacagc aacccgcgtg tagacctgaa tctgcaccct atcggtaatt ataaatatag tggtagtaat tcactcgaca tgtgtttcta tgatggatat agtacaaaca gtgatagcat ggtaataaag ttccaggatg ataatcctac caattcatct gaatataatc tttataagat agggggcact gaaaaattac catatgctgt ttcactgctt atgggaggaa aaatatttta tccagtgaat ggtcaatcat ttactatcaa tgacagtagt gtactcgaaa caaactggaa tcgagtaacc gcagttgcta tgccggaagt taatgttcca gtattatgct ggccagcaag attgctatta aatgctgatg taaatgctcc cgatgcagga cagtattcag gacagatata tataacattt acacccagtg tcgaaaattt atga Primers to amplify the segment for coating antigen in antibody titration ELISA RN-CS19-F: GTC ACC ATG GAT ATG ACA GGA TTG TTT TTG TAC (SEQ ID No. 118) RN-CS19-R: GGT GCG GCC GTT ATA TAT ATC TGT CCT GAA (SEQ ID No. 119) EaeH (SEQ ID No. 33) M S H Y K T G H K Q P R F R Y S V L A R C V A W A N I S V Q V L F P L A V T F T P V M A A R A Q H A V Q P R L S M G N T T V T A D N N V E K N V A S F A A N A G T F L S S Q P D S D A T R N F I T G M A T A K A N Q E I Q E W L G K Y G T A R V K L N V D K D F S L K D S S L E M L Y P I Y D T P T N M L F T Q G A I H R T D D R T Q S N I G F G W R H F S G N D W M A G V N T F I D H D L S R S H T R I G V G A E Y W R D Y L K L S A N G Y I R A S G W K K S P D I E D Y Q E R P A N G W D I R A E G Y L P A W P Q L G A S L M Y E Q Y Y G D E V G L F G K D K R Q K D P H A I S A E V T Y T P V Epitope (222-237): KKSPDIEDYQERPANG (SEQ ID No. 28) EaeH Nucleotide Sequence (SEQ ID No. 113) ATGTCACATTATAAAACAGGTCATAAACAACCACGATTTCGTTATTCAGTTCTGGCC CGCTGCGTGGCGT GGGCAAATATCTCTGTTCAGGTTCTTTTTCCACTCGCTGTCACCTTTACCCCAGTAAT GGCGGCACGTGC GCAGCATGCGGTTCAGCCACGGTTGAGCATGGGAAATACTACGGTAACTGCTGATA ATAACGTGGAGAAA AATGTCGCGTCGTTTGCCGCAAATGCCGGGACATTTTTAAGCAGTCAGCCAGATAGC GATGCGACACGTA ACTTTATTACCGGAATGGCCACAGCTAAAGCTAACCAGGAAATACAGGAGTGGCTC GGGAAATATGGTAC TGCGCGCGTCAAACTGAATGTCGATAAAGATTTCTCGCTGAAGGATTCTTCGCTGGA AATGCTTTATCCG ATTTATGATACGCCGACAAATATGTTGTTCACTCAGGGGGCAATACATCGTACAGAC GATCGTACTCAGT CAAATATTGGTTTTGGCTGGCGTCATTTTTCAGGAAATGACTGGATGGCGGGGGTGA ATACTTTTATCGA TCATGATTTATCCCGTAGTCATACCCGCATTGGTGTTGGTGCGGAATACTGGCGCGA TTATCTGAAACTG AGCGCCAATGGTTATATTCGGGCTTCTGGCTGGAAAAAATCGCCGGATATTGAGGAT TATCAGGAACGCC CGGCGAATGGCTGGGATATTCGTGCTGAGGGCTATTTACCCGCCTGGCCGCAGCTTG GCGCAAGCCTGAT GTATGAACAGTATTATGGCGATGAAGTCGGGCTGTTTGGTAAAGATAAGCGCCAGA AAGACCCGCATGCT ATTTCTGCCGAGGTGACCTATACGCCAGTGCC Primers to amplify the segment for coating antigen in antibody titration ELISA RN-EaEH-F: TTT ACC ATG GTA ATG GCG GCA CGT GCG CAG CAT (SEQ ID No. 120) RN-EaEH-R: TTT CCG GCC GTT ATC TTT ACC AAA CAG CCC (SEQ ID No. 121

Results and Conclusions:

Adult female Balb/C mice subcutaneously immunized with 40 μg tip MEFA-II, with 2 μg dmLT (double mutant LT, LT_(R192G/L211A)) adjuvant, one primary and two boosters at an interval of two weeks, developed antibody responses to CS12, CS14, CS17, CS19 and EaeH adhesins (FIG. 5). No antigen-specific antibody response was detected in the control mice.

FIG. 5 shows mouse serum IgG antibody titers (log₁₀), where mice immunized with adhesion tip MEFA-II developed anti-CS12, anti-CS14, anti-CS17, and anti-EaeH IgG antibodies. Each dot in FIG. 5 represented IgG titer from an individual mouse, where the bars indicate mean IgG titers.

ETEC field isolates expressing CS12, CS14, CS17, CS19 or EaeH, after incubated with serum of mice SC immunized with the adhesin tip MEFA-II, showed significant reduction in adherence to Caco-2 cells, compared to ETEC strains incubated with serum of the control mice. That suggested serum antibodies of the immunized mice were neutralizing, inhibited adherence of ETEC bacteria expressing any of these five adhesins.

TABLE 4 Results from mouse serum antibody in vitro adherence inhibition assays. Caco-2 cells (5 × 10⁵) were incubated with each ETEC bacteria strain expressing a different adhesin (in PBS), ETEC bacterial premixed with serum of the control mice, or ETEC bacteria premixed with serum of the mice immunized with adhesin tip MEFA-II. serum of serum of ETEC strains control mice immunized mice 3276, CS12/CS20/LT/STa (×10³) 227.6 ± 21.2  156 ± 17.1 p = 0.01 E7476A, CS14 (×10²) 91.8 ± 7.0 63.4 ± 6.3 p < 0.01 E20738A, CS17 (×10²) 137.2 ± 8.1  19.4 ± 3.0  p < 0.001 D526-1, CS19 (×10²) 92.2 ± 6.7 70.2 ± 9.2 p = 0.02 H10407-EaeH/EtpA/CFA/I/LT/STa 126.6 ± 32.4  67.2 ± 15.3 (×10²) p = 0.04

Example 3

The method described in Example 1 above will be completed for another embodiment of the present disclosure, a major subunit CFA MEFA-II utilizing the backbone of CS21 having epitopes for CS19, CS17, EtpA, CS14, CS7, CS12, and EaeH.

Materials and Methods

This CFA MEFA-II used CS21 major subunit LngA as the backbone, and has LngA surface-exposed and less antigenic epitopes substituted by the most antigenic epitopes from the major subunits of CS7, CS12, CS14, CS17, CS19, EtpA and EaeH adhesins. Therefore, CFA MEFA-II carried antigenic elements of 8 adhesins of enterotoxigenic Escherichia coli (ETEC), and is expected to induce antibodies against all these 8 ETEC adhesins (CS7, CS12, CS14, CS17, CS19, CS21, EtpA and EaeH).

The structure of CFA MEFA-II of Example 3 is shown in FIG. 6.

CFA MEFA-H amino acid sequence (SEQ ID No. 67)) MSLLEVIIVLGIIGTIAAGVVILAQRAFDSRAVTDLEAAPPEAGNYTVRVAMKDAYQRD GKYPDFVDPLSLTANTIKTDTSGIPAAQLVQLGKADTQGTAPEAGNYISGDFIAIGGAVE GEKSPRRGVKKGFAIELNGLTSGTAPSAGKYQEQCRSILGQVGAGSPVTRSDTTSWEYV AGSNGQANNNDASQVDMSVAASTTVLRSLGNKKSPDIEDYQERTADKILSTCTAQVNSI TLGSR CS7 Epitope: AGSPVTRSDTTS (SEQ ID No. 68) CS12 Epitope: GSNGQANNNDASQ (SEQ ID No. 69) CS14 Epitope: TSGTAPSAGKYQ (SEQ ID No. 70) CS17 Epitope: ADTQGTAPEAGNY (SEQ ID No. 71) CS19 Epitope: EAAPPEAGNY (SEQ ID No. 72) EtpA Epitope: VEGEKSPRRGV (SEQ ID No. 40) EaeH Epitope: KKSPDIEDYQER (SEQ ID No. 73) CFA MEFA-H DNA sequence (SEQ ID No. 74) ATGAGCCTGCTGGAAGTTATCATTGTTCTTGGCATTATCGGTACGATTGCAGCCGGT GTCGTGATTCTGGCTCAGCGTGCGTTTGATTCACGTGCTGTGACTGATTTAGAAGCT GCTCCGCCTGAGGCAGGTAATTACACAGTCCGCGTAGCAATGAAAGATGCTTATCA ACGTGATGGTAAATATCCAGATTTTGTGGACCCATTAAGCCTTACTGCAAATACAAT TAAAACTGATACAAGCGGAATACCTGCAGCACAGTTAGTTCAGCTTGGGAAAGCAG ACACACAAGGAACTGCGCCTGAGGCAGGCAATTAC(CS17Epitope)ATTTCTGGCGACT TTATCGCTATTGGCGGTGCTGTGGAGGGCGAAAAGTCCCCCCGTCGTGGCGTGAAAA AAGGTTTTGCTATCGAACTTAATGGATTAACATCCGGAACTGCACCAAGTGCAGGTA AGTATCAAGAGCAGTGCCGTTCTATTCTTGGGCAAGTTGGGGCTGGATCGCCGGTTA CACGTAGTGATACGACATCTTGGGAATATGTTGCTGGTAGTAACGGCCAAGCGAAT AACAATGATGCAAGCCAGGTAGATATGTCTGTGGCCGCCTCTACAACTGTTTTACGC TCTTTAGGTAACAAAAAATCGCCGGATATTGAGGATTATCAGGAACGCACTGCAGA CAAAATTCTAAGTACCTGTACTGCTCAGGTAAACTCAATTACTTTAGGTAGCCGTTA A CS7 major subunit (CsvA) (SEQ ID No. 75) Amino Acid sequence: MKKKLLIASVLAMATVSGSVLAAVTNGQLTFNWQGVVPSAPVTKNTWAFVNGLDIPFT PGTEQLNITLGADKGITARSVKPYDFFIVPVTGTVTAGSPVTRSDTTSMNSVKAFLSSEPV SNGFVGNKQLTLSTTAEAVTGQVAITLNGQPLKVGSANATTVAMDTNKKESHISIDMN AKASASDVAEGSAINFVAPVTFAVDI Epitope: 95-106 (12aa) Nucleotide sequence: (SEQ ID No. 76) ATGAAGAAAAAATTACTGATAGCTTCAGTATTAGCAATGGCAACCGTATCAGGTT CTGTTTTGGCTGCTGTTACAAATGGCCAGCTCACATTTAATTGGCAGGGAGTGGTTC CTTCTGCTCCCGTTACTAAAAATACGTGGGCTTTTGTGAACGGATTGGATATACCGTT TACTCCTGGTACTGAACAGTTGAATATCACCCTTGGTGCAGATAAAGGTATCACAGC CCGTTCGGTTAAGCCTTATGATTTTTTCATTGTTCCAGTTACTGGAACAGTAACTGCT GGATCGCCGGTTACACGTAGTGATACGACATCTATGAATAGTGTGAAAGCTTTTCTA TCAAGTGAACCCGTTTCTAATGGTTTTGTTGGCAACAAGCAGTTAACCCTGAGCACC ACAGCAGAAGCAGTTACGGGGCAAGTCGCAATCACTTTAAATGGTCAGCCGCTTAA AGTGGGGAGTGCTAATGCAACAACTGTTGCTATGGATACCAATAAAAAAGAGTCTC ATATTTCTATTGATATGAATGCCAAGGCAAGTGCTTCGGATGTGGCGGAGGGTTCA GCTATTAACTTTGTAGCTCCGGTAACATTTGCTGTTGATATTTAA Primers to amplify the gene segment as antibody titration ELISA coating antigen: CS7-CsvA-F: CGC CAT GGA AAT GGC AAC CGT ATC AG (NcoI) (SEQ ID No. 77) CS7-CsvA-R: TCG GCC GCT TTA CGC CAC ATC CGA AGC ACT T (EagI) (SEQ ID. No 78) CS12 major subunit (CswA) (SEQ ID No. 79) Amino acid sequence MRKITSLIMAVTLMNSAFAAIGSNGQANNNDASQAELHFTGKLTSSLCQVATSDVKKE IDLGELSKAALIASGRGPSQSFSVSLVNCDPTINTISYALQDKNGSVGNYLVNQSGDTMA KGVGVYIENNLNSPLKVDGSLNTVGVQKDGATALPDQVIPLTAYIGSTTPGAVADFATV TPGLVDANAVMTIRASAP Epitope: 23-35 (13aa) Nucleotide sequence (SEQ ID No. 80) ATGAGAAAAATTACGTCTCTGATCATGGCTGTTACTCTAATGAATAGCTCAGCCTT CGCTGCGATCGGTAGTAACGGCCAAGCGAATAACAATGATGCAAGCCAGGCTGAAT TGCATTTCACTGGTAAGTTGACTTCCAGTCTATGCCAGGTTGCTACATCAGATGTTAA AAAAGAAATTGATCTAGGGGAACTTAGCAAAGCTGCGTTAATTGCATCAGGTCGAG GGCCATCGCAGTCTTTTTCAGTTAGTTTGGTAAACTGTGACCCCACAATAAATACAA TTAGTTATGCTTTGCAGGATAAAAACGGCAGTGTAGGTAATTATCTTGTAAACCAGT CAGGTGACACGATGGCTAAGGGGGTTGGTGTCTATATTGAAAATAATTTAAATAGCC CTTTGAAGGTTGATGGTTCACTTAATACTGTTGGTGTTCAGAAAGATGGTGCTACG GCTTTACCTGATCAGGTTATTCCGTTGACTGCTTATATTGGTAGCACAACTCCGGGA GCTGTGGCCGATTTTGCTACAGTAACTCCTGGCTTAGTTGATGCAAATGCAGTAATG ACTATTCGTGCGAGCGCACCATAA Primers to amplify the subunit segment as antibody titration ELISA coating antigen: CA12-CswA-F: CATGCCATGGAAATGGCTGTTACTCTAATGAATAGCTCA (NcoI) (SEQ ID No. 81) CA12-CswA-R: GAGTCGGCCGCTTTATGAACACCAACAGTATTAAGTGAA (EagI) (SEQ ID No. 82) CS14 major subunit (CsuA) (SEQ ID No. 83) Amino acid sequence MKLKKTIGAMALSTIFVAVSASAVEKNITVTASVDPTIDILQANGSALPTAVDLTYLPGA KTFENYSVLTQIYTNDPSKGLDVRLVDTPKLTNILQPTSTIPLTVSWAGKTLSTSAQKIAV GDLGFGSTGTAGVSNSKELVIGATTSGTAPSAGKYQGVVSIVMTQSTDTAAPVP Epitope: 146-157 (12aa) Nucleotide sequence (SEQ ID No. 84) ATGAAATTAAAAAAAACTATTGGCGCAATGGCTCTGAGCACAATATTTGTAGCGG TGAGTGCTTCAGCAGTAGAGAAAAATATTACTGTGACAGCCAGTGTTGATCCTACTA TTGATATTCTTCAAGCAAATGGTTCTGCGCTACCGACAGCTGTAGATTTAACTTATCT ACCTGGTGCAAAAACTTTTGAAAATTACAGTGTTCTAACCCAGATTTACACAAATGA CCCTTCAAAAGGTTTAGATGTTCGACTGGTTGATACACCGAAACTTACAAATATTTT GCAACCGACATCTACCATTCCTCTTACTGTCTCATGGGCAGGGAGGACATTAAGTAC AAGTGCTCAGAAGATCGCAGTTGGCGATCTGGGTTTTGGTTCCACCGGAACGGCAG GTGTTTCGAATAGTAAAGAATTAGTAATTGGAGCAACTACATCCGGAACTGCACCA AGTGCAGGTAAGTATCAAGGCGTCGTTTCCATTGTAATGACTCAATCGACAAACT AA Primer to amplify the subunit the segment as antibody titration ELISA coating antigen: CA14-CsuA-F: CATGCCATGGAAATGGCTCTGAGCACAATATTTGTAG (NcoI) (SEA ID No. 85) CA14-CsuA-R: GAGTCGGCCGCTTTAGTTTGTCGATTGAGTCATTACAATGGA (EagI) (SEQ ID No. 86) CS17 major subunit gene (CsbA) (SEQ ID No. 87) Amino acid sequence MKLKKTIGAMAMATLFATMAASAVEKNITVRASVDPKLDLLQADGTSLPDSIALTYSS ASNNFEVYSLNTAIHTNDKSKGVVVKLSASPVLSNIMKPNSQIPMKVTLGGKTLNTTDT EFTVDTLNFGTSGVENVSSTQQLTIHADTQGTAPEAGNYQGIISLIMTQKT Epitope: 144-156 (13aa) Nucleotide sequence (SEQ ID No. 88) ATGAAACTGAAGAAAACAATTGGCGCAATGGCTATGGCGACTCTGTTTGCCACCA TGGCTGCCTCTGCAGTCGAAAAAAATATTACTGTGAGGGCAAGTGTTGACCCTAAAC TTGATCTTCTGCAAGCAGATGGAACTTCACTGCCGGACTCTATCGCATTAACCTATTC TTCGGCTTCAAATAATTTTGAAGTTTACTCTCTTAATACTGCTATTCATACAAATGAC AAAAGCAAGGGAGTTGTAGTGAAGCTGTCAGCTTCACCAGTTCTGTCCAATATTATG AAGCCAAACTCGCAAATTCCGATGAAAGTGACTTTGGGGGGGAAGACGCTGAATAC AACTGATACTGAGTTTACTGTTGATACTCTGAACTTTGGTACATCTGGTGTTGAAAA CGTTTCTTCCACTCAACAGCTTACGATTCATGCAGACACACAAGGAACTGCGCCTGA GGCAGGCAATTACCAAGGTATTATTTCTCTTATCATGACTCAAAAAACTTAA Primers to amplify the segment as antibody titration ELISA coating antigen: CA17-CsbA-F: CTAGCTAGCTAGATGGCTATGGCGACTCTGTTTGCCA (Nhe I) (SEQ ID No. 89) CA17-CsbA-R: GAGTCGGCCGCTTTATAATACCTTGGTAATTGCCTGCCTCA (EagI) (SEQ ID No. 90) CS19 major subunit (CsdA) (SEQ ID No. 91) Amino acid sequence: MKLKKTIGAMAMATLFATMAASAVEKNITVRASVDPKLDLLQADGTSLPDSIALTYSS ASNNFEVYSLNTAIHTNDKTKAVVVKLSAPAVLSNIMKPSSQIPMKVTLGGKTLSTADA EFAADTLNFGASGVENVSSVQQLTIHAEAAPPEAGNYQGVISLIMTQKT Epitope: 145-154 (10aa) Nucleotide sequence (SEQ ID No. 92) ATGAAACTGAAGAAAACAATTGGCGCAATGGCTATGGCGACTCTGTTTGCCACCA TGGCTGCCTCTGCAGTCGAAAAAAATATTACTGTGAGGGCAAGTGTTGACCCTAAAC TTGATCTTCTGCAAGCAGATGGAACTTCACTGCCGGACTCTATCGCATTAACCTATTC TTCGGCTTCAAATAATTTTGAAGTTTACTCTCTTAATACTGCTATTCATACAAATGAC AAAACCAAGGCAGTTGTAGTGAAGCTGTCAGCTCCAGCAGTTCTGTCCAATATTATG AAGCCAAGCTCGCAAATTCCGATGAAAGTGACTTTGGGGGGGAAGACGCTGAGTAC AGCTGATGCTGAGTTTGCTGCTGATACTCTGAACTTTGGTGCATCTGGTGTTGAAAA CGTTTCTTCCGTTCAACAGCTTACGATTCATGCAGAAGCTGCTCCGCCTGAGGCAGG TAATTACCAAGGTGTTATTTCTCTTATCATGACTCAAAAAACTTAA Primers to amplify the subunit segment as antibody titration ELISA coating antigen: CA19-CsdA-F: CTAGCTAGCTAGATGGCTATGGCGACTCTGTTTGCCA (Nhe I) (SEQ ID No. 93) CA19-CsdA-R: GAGTCGGCCGCTTTAGAGTCATGATAAGAGAAATAACAC (EagI) (SEQ ID No. 94) CS21 major subunit (LngA) (SEQ ID No. 95) Amino acid sequence MSLLEVIIVLGIIGTIAAGVVILAQRAFDSRAVTDLVTNTNTVRVAMKDAYQRDGKYPD FVDPLSLTANTIKTDTSGIPAAQLVQLGKITPDEVRNNISGDFIAIGGALTSNGAQVKKGF AIELNGLSQEQCRSILGQVGNNWEYVAIGTSASGSYAMTATGVDMSVAASTTVLRSLG NGGQTTLTADKILSTCTAQVNSITLGSR Epitope: 49-60 (12aa) 69-81 (13aa) Nucleotide sequence (SEQ ID No. 96) ATGAGCCTGCTGGAAGTTATCATTGTTCTTGGCATTATCGGTACGATTGCAGCCG GTGTCGTGATTCTGGCTCAGCGTGCGTTTGATTCACGTGCTGTGACTGATTTAGTAAC TAATACAAATACAGTCCGCGTAGCAATGAAAGATGCTTATCAACGTGATGGTAAAT ATCCAGATTTTGTGGACCCATTAAGCCTTACTGCAAATACAATTAAAACTGATACAA GCGGAATACCTGCAGCACAGTTAGTTCAGCTTGGGAAAATTACACCAGACGAAGTG CGTAATAACATTTCTGGCGACTTTATCGCTATTGGCGGTGCTTTAACTTCGAATGGTG CTCAAGTTAAAAAAGGTTTTGCTATCGAACTTAATGGATTAAGCCAAGAGCAGTGCC GTTCTATTCTTGGGCAAGTTGGGAATAACTGGGAATATGTTGCTATTGGTACTTCTGC GTCTGGTTCATATGCCATGACAGCAACTGGTGTAGATATGTCTGTGGCCGCCTCTAC AACTGTTTTACGCTCTTTAGGTAACGGTGGACAAACAACCTTGACTGCAGACAAAAT TCTAAGTACCTGTACTGCTCAGGTAAACTCAATTACTTTAGGTAGCCGTTAA Primers to amplify LngA gene the segment as antibody titration ELISA coating antigen: CA21-LngA-F: CATGCCATGGGCATGAGCCTGCTGGAAGTTATCATTGTT (NcoI) (SEQ ID No. 97) CA21-LngA-R: GAGTCGGCCGCTTTAACGGCTACCTAAAGTAATTGAG (EagI) (SEQ ID No. 98) EtpA adhesive subunit Amino acid sequence (SEQ ID No. 99) VSEITTGVGNAKATGSVEGEKSPRRGVRAMALSLLSGMMINIAHPAMSANLPTGGQIVA GSGSIQTPSGNQMNIHQNSQNMVANWNSFDIGKGNTVQFDQPSSSAVALNRVVGGGES QIMGNLKANGQVFLVNPNGVLFGEGASVSTSGFVASTRDIKNDDFMNRRYTFSGGQKA GAAIVNQGELTTNAGGYIVLAADRVSNSGTIRTPGGKTVLAA Epitope: 17-27 (11aa) Nucleotide sequence (SEQ ID No. 100) ATGAACCGTATATATAAACTGAAGTTTGACAAACGCCGCAACGAACTGGTGGTGG TGAGTGAAATCACCACCGGCGTGGGTAATGCAAAAGCCACGGGCAGCGTGGAGGGC GAAAAGTCCCCCCGTCGTGGCGTGCGCGCCATGGCGCTGAGCCTGCTGTCGGGTATG ATGATAATGGCCCATCCGGCGATGTCAGCAAACCTGCCGACCGGTGGCCAGATTGT GGCAGGTTCAGGCAGTATCCAGACGCCTTCCGGCAACCAGATGAATATTCATCAGA ACAGCCAGAACATGGTGGCCAACTGGAACAGCTTTGACATTGGTAAAGGAAATACG GTGCAGTTTGACCAGCCCAGCAGCAGTGCGGTGGCGCTGAACCGTGTTGTGGGTGG CGGTGAATCGCAGATTATGGGTAACCTGAAGGCGAATGGTCAGGTGTTCCTGGTTAA CCCGAACGGCGTGCTGTTTGGTGAGGGGGCCAGTGTCAGCACGTCAGGTTTTGTGGC ATCGACCCGCGACATTAAAAACGACGACTTCATGAACCGTCGTTACACCTTCAGCGG CGGACAGAAAGCCGGGGCAGCGATTGTGAACCAGGGGGAACTGACCACAAATGCC GGTGGCTATATTGTGCTGGCAGCAGACAGGGTCAGCAACAGTGGCACCATCCGTAC GCCGGGCGGCAAGACCGTCCTGGCGGCCAGCGAGCGCATCACGCTGCAGCTGGATA ATGGTGGCCTGATGTCCGTGCAGGTGACAGGAGATGTGGTTAATGCCCTGGTGGAA AACCGCGGTCTGGTCAGTGCCCGGGATGGTCAGGTGTACCTGACCGCACTTGGCCG GGGTATGCTGATGAACACGGTACTGAACGTGAGCGGGGTGGTGGAAGCCAGCGGTA TGCACCGTCAGGACGGTAACATTGTACTGGACGGTGGCGACAGTGGTGTGGTGCAC CTGAGTGGTACCCTGCAGGCGGACAATGCGTCCGGTCAGGGTGGTAAGGTTGTCGT GCAGGGTAAGAATATTCTGCTGGACAAGGGCAGCAACATCACAGCAACCGGTGGTC AGGGCGGCGGTGAAGTGTATGTCGGTGGCGGCTGGCAGGGTAAGGACAGCAACATC CGTAATGCGGACAAGGTGGTGATGCAGGGCGGCGCCCGCATTGACGTTTCTGCAAC GCAGCAGGGTAACGGCGGTACGGCTGTGCTGTGGTCAGACAGCTACACCAACTTCC ATGGTCAGATTAGCGCGAAGGGCGGTGAGACCGGCGGTAACGGTGGTCGGGTGGA GACCTCTTCGCACGGTAACCTGCAGGCATTTGGTACGGTCAGTGCATCCGCGAAGAA AGGCAAGGCGGGTAACTGGCTGCTGGACTCGGCGGATATCACCATTGTGAATGGTA GCAATGTTAGCAAAACTGAGACGACTCAATCGCCGCCGCACACGCAATTTGCACCC ACCGCTGCGGGCTCTGCGGTCAGCAATACCAGTATCAACAACAGGCTGAACAACGG GACCAGTGTCACTATTCTGACCCATCGCACAAGAACAGGCACAGCTCAGGGCGGGA ATATTACCGTTAATGCGGCAATTAACAAAAGCAACGGAAGTGATGTCAACCTGACG CTGCAGGCTGGCGGCAACATCACGGTAAACAACAGCATCACGTCCACCGAGGGTAA GCTGAATGTTAATCTGTCGGGCGCCAGGACCAGCAATGGCAGTATCACCATTAGCA ATAACGCCAATATAACGACCAATGGTGGGGATATAACTGTTGGGACGACAAATACT TCAAACCGTGTGAATATATCTATTAATAACACTACCCTGAATGCGTCAAATGGCAAC ATCCAGTTGACCGGGACCGGGACCGATAGCGGGATTCTGTTTGCTGGCAACAACAG GCTGACGGCCAGTAACATTGCTCTTACCGGGAACAGTACGAGTGGGAATGCCATCA ACCTTACAGGCACTGCCACGCTGAATGCCACGAATAACATTACTCTTACCGGGAGCA GTACGAGTGGGAATGCCATCAACCTTAAAGGCAACAACACGCTGACGGCCAGTAAC ATTACTCTTACCGGGGAAAGTACGAGTGGGAATGCCATCAACCTTACAGACACTAC AGGCACTACCACGCTGAATGCCACGAATAACATCACTATGCAGGGGACCCGTGTTC AGATTAAACACTCCAACATCACCGCGGGCAACTTTGCGCTGAATGCGACAGTGGCC GGCTCTGAAATCAGCAATACCACGCTGACGGCCACCAACAACATCAACCTGGCGGC TAAGACGAACAGTGCGAGCTCTGGTGTTTACCTGAAAGATGCAAGAATTACATCCA CCAATGGCAGTATCACGGCTAACGGTACTGCCACAGCAAACGGCAAGGCCACGCAT CTGGACGGCAACGTCACCCTGAATGCGTCAAATGGCAGAATCAAGTTGACCGGGAA CGGGCACGGTAGCGCCTCCGGGATTCTGTTTGCTGGCAACAACAGGCTGACGGCCA GTAACATTGCTCTTACCGGGAACAGTACGAGTGGGAATGCCATCAACCTTACAGGC ACTGCCACGCTGAATGCCACGAATGACATTACTCTTACCGGGAGCAGTACGAGTGG GAATGCCATCAACCTTACAGGCACTGCCACGCTGAATGCCACGAATAACATTACTCT TACCGGGAGCAGTACGAGTGGGAATGC CATCAACCTTAAAGGCAACAACACGCTGACGGCCAGTAACATTACTCTTACCGGGG AAAGTACGAGTGGGAATGCCATCAACCTTACAGACACTACAGGCACTACCACGCTG AATGCCACGAATAACATCACTATGCAGGGGACCCGTGTTCAGATTAAACACTCCAA CATCACCGCGGGCAACTTTGCGCTGAATGCGACAGTGGCCGGCTCTGAAATCAGCA ATACCACGCTGACGGCCACCAACAACATCAACCTGGCGGCTAAGACGAACAGTGCG AGCTCTGGTGTTTACCTGAAAGATGCAAGAATTACATCCACCAATGGCAGTATCACG GCTAACGGTACTGCCACAGCAAACGGCAAGGCCACGCATCTGGACGGCAACGTCAC CCTGAATGCGTCAAATGGCAGAATCAAGTTGACCGGGAACGGGCACGGTAGCGCCT CCGGGATTCTGTTTGCTGGCAACAACAGGCTGACGGCCAGTAACATTGCTCTTACCG GGAACAGTACGAGTGGGAATGCCATCAACCTTACAGGCACTGCCACGCTGAATG CCACGAATGACATTACTCTTACCGGGAGCAGTACGAGTGGGAATGCCATCAACCTTA CAGGCACTGCCACGCTGAATGCCACGAATAACATTACTCTTACCGGGAGCAGTACG AGTGGGAATGCCATCAACCTTAAAGGCAACAACACGCTGACGGCCAGTAACATTAC TCTTACCGGGGAAAGTACGAGTGGGAATGCCATCAACCTTACAGACACTACAGGCA CTACCACGCTGAATGCCACGAATAACATCACTATGCAGGGGACCCGTGTTCAGATTA AACACTCCAACATCACCGCGGGCAACTTTGCGCTGAATGCGACAGTGGCCGGCTCT GAAATCAGCAATACCACGCTGACGGCCACCAACAACATCAACCTGGCGGCTAAGAC GAACAGTGCGAGCTCTGGTGTTTACCTGAAAGATGCAAGAATTACATCCACCAATG GCAGTATCACGGCTAACGGTACTGCCACAGCAAACGGCAAGGCCACGCATCTGGAC GGCAACGTCACCCTGAATGCGTCAAATGGCAGAATCAAGTTGACCGGGAACGGG CACGGTAGCGCCTCCGGGATTCTGTTTGCTGGCAACAACAGGCTGACGGCCAGTAA CATTGCTCTTACCGGGAACAGTACGAGTGGGAATGCCATCAACCTTACAGGCACTGC CACGCTGAATGCCACGAATGACATTACTCTTACCGGGAGCAGTACGAGTGGGAATG CCATCAACCTTACAGGCACTGCCACGCTGAATGCCACGAATAACATTACTCTTACCG GGAGCAGTACGAGTGGGAATGCCATCAACCTTAAAGGCAACAACACGCTGACGGCC AGTAACATTACTCTTACCGGGGAAAGTACGAGTGGGAATGCCATCAACCTTACAGA CACTACAGGCACTACCACGCTGAATGCCACGAATAACATCACTATGCAGGGGACCC GTGTTCAGATTAAACACTCCAACATCACCGCGGGCAACTTTGCGCTGAATGCGACAG TGGCCGGCTCTGAAATCAGCAATACCACGCTGACGGCCACCAACAACATCAACCTG GCGGCTAAGACGAACAGTGCGAGCTCTGGTGTTTACCTGAAAGATGCAAGAATTAC ATCCACTAATGGCAGTATCACGACTAACGGTACTGCCACAGCAAACGGCAAGGCCA CGCATCTGGACGGCAACGTCACCCTGAATGCGTCAAATGGCAGAATCAAGTTGACC GGGAACGGGCACGGTAGCGCCTCCGGGATTCTGTTTGCTGGCAACAACAGGCTGAC GGCCAGTAACATTGCTCTTACCGGGAACAGTACGAGTGGGAATGCCATCAACCTTAC AGGCACTGCCACGCTGAATGCCACGAATGACATTACTCTTACCGGGAGCAGTACGA GTGGGAATGCCATCAACCTTACAGGCACTGCCACGCTGAATGCCACGAATAACATT ACTCTTACCGGGAGCAGTACGAGTGGGAATGCCATCAACCTTAAAGGCAACAACAC GCTGACGGCCAGTAACATTACTCTTACCGGGGAAAGTACGAGTGGGAATGCCATCA ACCTTACAGACACTACAGGCACTACCAC GCTGAATGCCACGAATAACATCACTATGCAGGGGACCCGTGTTCAGATTAAACACTC CAACATCACCGCGGGCAACTTTGCGCTGAATGCGACAGTGGCCGGCTCTGAAATCA GCAATACCACGCTGACGGCCACCAACAACATCAACCTGGCGGCTAAGACGAACAGT GCGAGCTCTGGTGTTTACCTGAAAGATGCAAGAATTACATCCACCAATGGCAGTATC ACGGCTAACGGTACTGCCCCAGCAAACGACAATGCCACGTATCTGGACGGCAACGT CACCCTGAATGCGTCAAATGGCAGCATCAAGTTGACCGGGAACGGGAACGGTAGCA CCTCCGGGATTCTGTTTGCTGGCAACAACACGCTGACGGCCAGTAACATTACTCTTA CCGGGAACAGTGAGGTGTACTGGCAATAG Primers to amplify the segment as antibody titration ELISA coating antigen: EtpA-F: CTAGCTAGCTAGATGAACCGTATATATAAACTG (NheI) (SEQ ID No. 101) EtpA-R: GAGTCGGCCGCTTTATAATCTGACCATGGAAGTTGGTGTA(Eag I) (SEQ ID No. 102) EaeH subunit Amino acid sequence: (SEQ ID No. 103) MSHYKTGHKQPRFRYSVLARCVAWANISVQVLFPLAVTFTPVMAARAQHAVQPRLSM GNTTVTADNNVEKNVASFAANAGTFLSSQPDSDATRNFITGMATAKANQEIQEWLGKY GTARVKLNVDKDFSLKDSSLEMLYPIYDTPTNMLFTQGAIHRTDDRTQSNIGFGWRHFS GNDWMAGVNTFIDHDLSRSHTRIGVGAEYWRDYLKLSANGYIRASGWKKSPDIEDYQE RPANGWDIRAEGYLPAWPQLGASLMYEQYYGDEVGLFGKDKRQKDPHAISAEVTYTP V Epitope: 222-233 (12aa) Nucleotide sequence (SEQ ID No. 104) ATGTCACATTATAAAACAGGTCATAAACAACCACGATTTCGTTATTCAGTTCTGGCCCG CTGCGTGGCGTGGGCAAATATCTCTGTTCAGGTTCTTTTTCCACTCGCTGTCACCTTTA CCCCAGTAATGGCGGCACGTGCGCAGCATGCGGTTCAGCCACGGTTGAGCATGGGA AATACTACGGTAACTGCTGATAATAACGTGGAGAAAAATGTCGCGTCGTTTGCCGCA AATGCCGGGACATTTTTAAGCAGTCAGCCAGATAGCGATGCGACACGTAACTTTATTA CCGGAATGGCCACAGCTAAAGCTAACCAGGAAATACAGGAGTGGCTCGGGAAATATG GTACTGCGCGCGTCAAACTGAATGTCGATAAAGATTTCTCGCTGAAGGATTCTTCGCT GGAAATGCTTTATCCGATTTATGATACGCCGACAAATATGTTGTTCACTCAGGGGGCAA TACATCGTACAGACGATCGTACTCAGTCAAATATTGGTTTTGGCTGGCGTCATTTTTCA GGAAATGACTGGATGGCGGGGGTGAATACTTTTATCGATCATGATTTATCCCGTAGTCA TACCCGCATTGGTGTTGGTGCGGAATACTGGCGCGATTATCTGAAACTGAGCGCCAAT GGTTATATTCGGGCTTCTGGCTGGAAAAAATCGCCGGATATTGAGGATTATCAGGAA CGCCCGGCGAATGGCTGGGATATTCGTGCTGAGGGCTATTTACCCGCCTGGCCGCAGC TTGGCGCAAGCCTGATGTATGAACAGTATTATGGCGATGAAGTCGGGCTGTTTGGTAA AGATAAGCGCCAGAAAGACCCGCATGCTATTTCTGCCGAGGTGACCTATACGCCAGT GCCTCTTCTGACACTGAGCGCCGGGCATAAGCAGGGCAAGAGTGGTGAGAATGACA CTCGCTTTGGCCTGGAAGTTAATTATCGGATTGGCGAACCTCTGGCGAAACAACTC GATACAGACAGCATTCGCGAGCGTCGGGTACTGGCAGGCAGCCGCTATGACCTGGTT GAGCGTAATAACAACATCGTTCTTGAGTACCGCAAATCTGAAGTGATCCGTATTGCTC TGCCTGAACGTATTGAAGGTAAGGGTGGTCAGACACTTTCCCTGGGGCTTGTGGTCA GCAAAGCAACTCACGGACTGAAAAATGTGCAGTGGGAAGCGCCGTCATTACTGGCT GAGGGTGGCAAAATTACCGGTCAGGGTAGTCAGTGGCAAGTAACGCTCCCGGCTTAT CGTCCAGGCAAAGACAATTATTATGCGATTTCTGCGGTTGCCTACGATAACAAAGGCA ATGCCTCAAAACGCGTGCAGACAGAGGTGGTCATTACCGGAGCAGGTATGAGCGCCG ATCGCACGGCGTTAACGCTTGACGGTCAGAGCCGTATTCAAATGCTTGCTAACGGTAA TGAGCAAAGACCGCTGGTGCTGTCTCTGCGCGACGCCGAGGGGCAGCCAGTCACGG GCATGAAAGATCAGATCAAGACTGAACTAGCCTTCAAACCGGCTGGAAATATTGTGA CTCGTTCCCTGAAGGCCACTAAATCACAGGCAAAGCCAACACTGGGTGAGTTCACCG AAACTGAAGCAGGGGTGTATCAGTCTGTCTTTACTACCGGAACGCAGTCAGGTGAGG CAACGATTACTGTTAGCGTTGATGGCATGAGCAAAACCGTCACTGCAGAACTGCGGG CCACGATGATGGATGTGGCAAACTCCACCCTGAGCGCTAACGAGCCGTCAGGTGATG TGGTTGCTGATGGTCAGCAAGCCTATACGTTGACGTTGACTGCGGTGGACTCCGAGG GTAATCCGGTGACGGGAGAAGCCAGCCGCTTGCGATTTGTTCCGCAAGACACTAATG GTGTAACCGTTGGTGCCATTTCGGAAATAAAACCAGGCGTTTACAGCGCCACGGTTTC TTCGACCCGTGCCGGAAACGTTGTTGTGCGTGCTTTCAGCGAGCAGTATCAGCTGGG CACATTACAACAAACGCTGAAGTTTGTTGCCGGTCCGCTTGATGCAGCACATTCGTCC ATCCCCCTGAATCCTGATAAACCGGTGGTTGGCGGTACAGTTACGGCAATCTGGACGG CAAAAGATGCCTATGACAACCCTGTGACCAGCCTCACGCCGGAAGCGCCGTCATTAG CGGGTGCCGCTGCTGTAGGTTCTACGGCATCTGGCTGGACAAATAATGGTGATGGGAC GTGGACTGCGCAGATTACTCTCGGCTCTACGGCGGGTGAATTAGAAGTTATGCCGAA GCTAAATGGACAGGATGCGGCAGCAAATGCGGCAAAAGTAACCGTGGTGGCTGATGC GTTATCTTCAAACCAGTCGAAAGTCTCTGTCGCAGAAGATCACGTAAAAGCCGGCGA AAGCACAACCGTGACGCTTATTGCAAAAGATGCACATGGCAACACTATCAGTGGTCT TTCGTTGTCGGCAAGTTTGACGGGGACCGCCTCTGAAGGGGCGACCGTTTCCAGTTG GACCGAAAAAGGTGACTGTTCCTATGTTGCTACGTTAACTACAGGCGGAAAGACGGG CGAGCTTCGTGTCATGCCGCTCTTCAACGGCCAGCCAGCAGCCACCGAAGCCGCGCA GTTGACGGTCATCGCCGGAGAGATGTCATCAGCGAACTCTACGCTTGTTGCGGACAAT AAGGCTCCGACCGTCAAAATGACGACGGAACTCACCTTCACCGTGAAGGATGCGTAC GGGAACCCGGTCACCGGGCTGAAGCCAGATGCACCAGTGTTTAGCGGTGCCGCCAG CACGGGGAGTGAGCGTCCTTCAGCAGGAAACTGGACAGAGAAAGGTAATGGGGTCT ACGTGGCGACCTTAACGCTGGGATCTGCCGCGGGTCAGTTGTCTGTGATGCCGCGAG TGAACGGCCAAAATGCCGTTGCTCAGCCACTGGTGCTGAACGTTGCAGGTGACGCAT CTAAGGCTGAGATTCGTGATATGACAGTGAAGGTTAATAACCAACTGGCTAATGGACA GTCTGCTAACCAGATAACCCTGACCGTCGTGGACAGCTATGGTAACCCGTTGCAGGG GCAAGAAGTTACGCTGACTTTACCGCAGGGTGTGACCAGCAAGACGGGGAATACAGT AACAACCAATGCGGCAGGGAAAGTGGACATTGAGCTTATGTCAACGGTTGCGGGGG AACACAGCATCACGGCCTCAGTGAATAATGCTCAGAAGACGGTTACGGTGAAATTCA AGGCGGATTTCAGTACCGGTCAGGCGACCCTGGAGGTTGATGGCAGCACGCCAAAA GTGGCAAACGACAATGATGCCTTTACGCTGACGGCAACGGTTAAGGATCAATACGGC AACCTTCTGCCTGGCGCTGTGGTCGTCTTTAATCTGCCTCGGGGCGTCAAACCGCTTG CAGACGGTAATATCATGGTGAACGCCGACAAGGAGGGTAAAGCGGAACTGAAAGTG GTCTCCGTGACTGCCGGAACGTATGAGATCACGGCGTCGGCAGGAAATGACCAGCCT TCGAATGCGCAGTCTGTAACGTTTGTGGCCGATAAGACTACGGCGACCATCTCCAGTA TTGAGGTGATTGGCAACCGTGCAGTGGCGGATGGCAAAACCAAACAGACGTATAAA GTTACGGTGACTGATGCCAATAACAACCTGTTGAAGGATAGCGACGTGACGCTGACT GCCAGCTCGGAAAATTTAGTTCTGGATCCTAAAGGGACGGCGAAAACTAATGAGCAA GGACAGGCTGTTTTCACCGGCTCTACCACTATCGCAGCGACATATACACTCACGGCGA AAGTGGAACAGGCCAACGGTCAGGTATCGACGAAAACTGCTGAATCTAAATTCGTCG CGGATGATAAAAACGCGGTGCTCGCCGCATCTCCAGAACGTGTAGATTCTCTGGTGG CGGACGGGAAGACTACTGCAACAATGACGGTTACCCTGATGGCGGGAGTCAATCCCG TAGGAGGAAGTATGTGGGTCGACATTGAGGCTCCGGAAGGAGTGACGGAGAAGGAT TATCAATTCCTGCCGTCGAAGGCTGACCATTTCTCAGGTGGGAAAATCACGCGTACAT TTAGTACCAGCAAGCCAGGTGTCTATACGTTCACATTCAACGCACTGACGTATGGCGG GTACGAAATGACGCCTGTGAAGGTGACAATTAACGCCGTTGCTGCAGAGACTGAAAA TGGCGAGGAGGAGATGCCATAA Primers to amplify the segment as antibody titration ELISA coating antigen EaeH-F: CATGCCATGGAAATGGGAAATACTACGGTAACTGC (NcoI) (SEQ ID No. 105) EaeH-R: GAGTCGGCCGCATTAGGTTCGCCAATCCGATAATTAACTT(EagI) (SEQ ID No. 106)

Adult female mice, 10 per group, were subcutaneously (SC) immunized with 40 μg recombinant CFA MEFA-II protein, with 2 μg dmLT adjuvant. Immunized mice received two boosters in an interval of two weeks. Two weeks after the final booster, mice were anesthetized and exsanguinated. A group of 10 mice without immunization were used as the control. Mouse serum samples were used for antibody titration and antibody adherence inhibition assay.

Antibody titration ELISAs using recombinant subunit proteins as coating antigens (400 ng per well) was performed. Serum samples from each mouse from the immunized group were detected great titers of anti-CS7, -CS12, -CS14, -CS17, -CS19, -CS21, -EaeH and anti-EtpA IgG antibodies.

Results and Conclusions

All mice in the immunization group developed antibodies specific to CS7, CS12, CS14, CS17, CS19, CS21, EaeH and EtpA. No antigen-specific antibodies were detected from serum samples of the control mice. Moreover, serum samples from the mice SC immunized with CFA MEFA-II showed antibody neutralization activities. ETEC bacteria expressing CS7, CS12, CS14, CS17, CS19, CS21, EaeH, or EtpA adhesin, after incubated with the serum samples of the immunized mice, showed a significant reduction in adherence to Caco-2 cells, compared the bacteria incubated with the serum samples of the control mice. These results are show in FIG. 7, where the IgG antibody titers are illustrated for each mouse, where each dot represented a IgG titer from an individual mouse and the bars indicated mean titers.

TABLE 5 Mouse serum antibody in vitro adherence inhibition assay results. Caco-2cells (5 × 10⁵) were incubated with each ETEC bacteria strain expressing a different adhesion (in PBS), ETEC bacteria premixed with serum of the control mice, or ETEC bacteria premixed with serum of the mice immunized with CFA MEFA-II serum of serum of control immunized E.coli strains PBS mice mice H10407- 136.8 ± 5.6  130.8 ± 4.4  44 ± 2.7 EaeH/EtpA/CFA/I/LT/STa p < 0.001 (×10²) JF2327, CS7/LT (×10³) 77.2 ± 3.1   75 ± 3.0 30.8 ± 2.3 p < 0.001 JF2363, CS7/EatA/LT 105.6 ± 5.1  100.8 ± 4.3 33.2 ± 1.9 (×10³) p < 0.001 3276, CS12/CS20/LT/STa  107 ± 6.3  100 ± 5.0 31.4 ± 2.6 (×10³) p < 0.001 3309,  145 ± 9.4  140 ± 7.1  37 ± 2.9 CS12/CS20/CS21/LT/STa p < 0.001 (×10³) E7476A, CS14 (×10²) 131.8 ± 3.1  127.4 ± 7.7 63.8 ± 3.2 p < 0.001 JF2125, CS14/EtpA/LT 75.4 ± 2.3  73.8 ± 5.5 15.6 ± 1.5 (×10²) p < 0.001 E20738A, CS17 (×10²) 117.8 ± 8.0    115 ± 10.8 50.4 ± 3.2 p < 0.001 JF2350, 219.8 ± 9.2  208.4 ± 9.6 133.2 ± 5.0  CS17/EtpA/EatA/LT p < 0.001 (×10²) D526-1, CS19 (×10²) 182.4 ± 10.4 179.4 ± 8.7  61 ± 3.4 p < 0.001 JF2101, 45.6 ± 3.0  40.6 ± 1.8 14.8 ± 1.5 CS21/EtpA/EatA/STa p < 0.001 (×10²) JF2318, EtpA/STa (×10²) 91.8 ± 5.8  87.2 ± 4.5 33.4 ± 3.5 p < 0.001 JF1389,  43 ± 2.3   41 ± 4.4 18.2 ± 2.6 EtpA/CS5/CS6/LT/STa p < 0.001 (×10²) 

1. A multiepitope fusion antigen selected from the group consisting of a multiepitope fusion antigen comprising a CfaE backbone having 3 or more different epitopes therein, selected from the group consisting of CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS12, CS14, CS17, CS19, CS21, EtpA, and EaeH, a multiepitope fusion antigen comprising a CS14 backbone having 3 or more different epitopes therein, selected from the group consisting of CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS12, CS14, CS17, CS19, CS21, EtpA, and EaeH, a major subunit CFA multiepitope fusion antigen comprising a CS21 backbone having 3 or more different epitopes therein, selected from the group consisting of CS7, CS12, CS14, CS17, CS19, CS21, EtpA, and EaeH, and any combination thereof.
 2. (canceled)
 3. (canceled)
 4. An immunogenic composition comprising the multiepitope fusion antigen of claim
 1. 5. (canceled)
 6. (canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled)
 11. A method of reducing the clinical signs of Enterotoxigenic Escherichia coli comprising the steps of administering the multiepitope fusion antigen of claim
 1. 12. (canceled)
 13. (canceled)
 14. (canceled)
 15. (canceled)
 16. (canceled)
 17. (canceled)
 18. The multiepitope fusion antigen of claim 1, wherein the multiepitope fusion antigen comprises a CfaE backbone comprising the epitopes of CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21, and EtpA.
 19. The multiepitope fusion antigen of claim 1, wherein the multiepitope fusion antigen comprises a CS14 backbone comprising the epitopes of CS12, CS17, CS19, and EaeH.
 20. The subunit CFA multiepitope fusion antigen of claim 1, wherein the major subunit CFA multiepitope fusion antigen comprises a CS21 backbone comprising the epitopes of CS7, CS12, CS14, CS17, CS19, EtpA, and EaeH.
 21. The multiepitope fusion antigen of claim 1, further comprising an additional element selected from the group consisting of heat liable toxin (LT), heat stable toxin (STa), an epitope from adhesin of enteroaggregative E. coli (EAEC), epitopes of cholera, epitopes of rotavirus, and any combination thereof.
 22. (canceled)
 23. (canceled)
 24. The multiepitope fusion antigen of claim 1, further comprising an additional element selected from the group consisting of a pharmaceutically acceptable vehicle, adjuvant, carrier and any combination thereof.
 25. (canceled)
 26. (canceled)
 27. The method of claim 11, wherein the administering step comprises intradermal, intramuscular, or subcutaneous administration.
 28. (canceled)
 29. (canceled)
 30. A method of reducing the adhesion of ETEC to the gut comprising the steps of administering the multiepitope fusion antigen of claim
 1. 31. (canceled)
 32. (canceled) 